Published in the June 2016 issue of Nature, the study found that tetrahydrocannabinol (THC), the main psychoactive compound in marijuana, and other active cannabis compounds could block the progression of the disease.

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Alzheimer’s disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and eventually the ability to carry out the simplest tasks. In most people with Alzheimer’s, symptoms first appear in their mid-60s. Estimates vary, but experts suggest that more than 5 million Americans may have Alzheimer’s.

Alzheimer's disease is currently ranked as the sixth leading cause of death in the United States, but recent estimates indicate that the disorder may rank third, just behind heart disease and cancer, as a cause of death for older people. 

 

ALZHEIMER'S DISEASE and cannabis studies complete

1997 - Study - Effects of dronabinol on anorexia and disturbed behavior in patients with Alzheimer's disease.

2003 - Study ~ Anandamide and noladin ether prevent neurotoxicity of the human amyloid-beta peptide.

2003 - Patent ~ US Patent 6630507 - Cannabinoids as antioxidants and neuroprotectants.

2003 - Study - Open-label study of Dronabinol in the treatment of refractory agitation in Alzheimer's disease.

2003 - Study - Safety and efficacy of Dronabinol in the treatment of agitation in patients with Alzheimer's disease with anorexia.

2003 - Study ~ Cannabinoid CB2 Receptors and Fatty Acid Amide Hydrolase Are Selectively Overexpressed in Neuritic Plaque-Associated Glia in Alzheimer's Disease Brains.

2004 - Study ~ Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on β-amyloid-induced toxicity in PC12 cells.

2005 - Study - Prevention of Alzheimer's Disease Pathology by Cannabinoids.

2005 - Study ~ Early age-related cognitive impairment in mice lacking cannabinoid CB1 receptors.

2005 - Study ~ Stimulation of cannabinoid receptor 2 (CB2) suppresses microglial activation.

2005 - Study ~ Avoidance of Abeta[(25-35)] / (H(2)O(2)) -induced apoptosis in lymphocytes by the cannabinoid agonists CP55,940 and JWH-015 via receptor-independent and PI3K-dependent mechanisms: role of NF-kappaB and p53.

2005 - Study ~ Phosphorylated amyloid-beta: the toxic intermediate in alzheimer's disease neurodegeneration.

2005 - Study ~ Cannabinoid control of motor function at the basal ganglia.

2005 - News ~ Marijuana Ingredient May Stall Decline From Alzheimer's.

2005 - News - MARIJUANA SLOWS ALZHEIMER'S DECLINE.

2005 - News - Marijuana may block Alzheimer's.

2005 - News - Cannabinoids reduce the progression of Alzheimer's disease in animals.

2005 - News ~ Research shows preventive effects of cannabinoids on Alzheimer's disease.

2005 - News ~ Marijuana Slows Alzheimer's Decline.

2005 - News ~ Pass the Doobie, pops.

2006 - Study ~ Delta-9-tetrahydrocannabinol for nighttime agitation in severe dementia.

2006 - Study ~ Endocannabinoids and beta-amyloid-induced neurotoxicity in vivo: effect of pharmacological elevation of endocannabinoid levels.

2006 - Study ~ Cannabidiol inhibits inducible nitric oxide synthase protein expression and nitric oxide production in beta-amyloid stimulated PC12 neurons through p38 MAP kinase and NF-kappaB involvement.

2006 - Study ~ The marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through Wnt/beta-catenin pathway rescue in PC12 cells.

2006 - Study ~ CB1 receptor selective activation inhibits beta-amyloid-induced iNOS protein expression in C6 cells and subsequently blunts tau protein hyperphosphorylation in co-cultured neurons.

2006 - Study - Molecular Link between the Active Component of Marijuana and Alzheimer's Disease Pathology.

2006 - News - Marijuana's Active Ingredient Shown to Inhibit Primary Marker of Alzheimer's Disease.

2006 - News - THC inhibits primary marker of Alzheimer's disease.

2006 - News ~ Marijuana Compound Efficient Against Alzheimer's Disease.

2006 - News ~ Marijuana's Active Ingredient May Slow Progression Of Alzheimer's Disease.

2006 - News ~ Marijuana may help stave off Alzheimer’s.

2006 - News ~ Marijuana May Slow Alzheimer's.

2006 - News ~ Pot-Like Compound May Slow Alzheimer's.

2006 - News ~ Latest Buzz: Marijuana May Slow Progression Of Alzheimer's Disease.

2007 - Study - Cannabidiol in vivo blunts β-amyloid induced neuroinflammation by suppressing IL-1β and iNOS expression.

2007 - Study - Alzheimer's disease; taking the edge off with cannabinoids?

2007 - Study - Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation.

2007 - Study ~ Opposing control of cannabinoid receptor stimulation on amyloid-beta-induced reactive gliosis: in vitro and in vivo evidence.

2007 - Study ~ The endocannabinoid system in targeting inflammatory neurodegenerative diseases.

2008 - Study - Cannabinoid receptor stimulation is anti-inflammatory and improves memory in old rats.

2008 - Study - Inflammation and aging: can endocannabinoids help?

2008 - Study ~ Cannabinoid CB2 receptors in human brain inflammation.

2008 - Study ~ Cannabinoids as Therapeutic Agents for Ablating Neuroinflammatory Disease.

2008 - Study ~ Amyloid precursor protein 96-110 and beta-amyloid 1-42 elicit developmental anomalies in sea urchin embryos and larvae that are alleviated by neurotransmitter analogs for acetylcholine, serotonin and cannabinoids.

2008 - Study ~ Role of CB2 receptors in neuroprotective effects of cannabinoids.

2008 - Study ~ The role of the endocannabinoid system in Alzheimer's disease: facts and hypotheses.

2008 - News - Pot joins the fight against Alzheimer's, memory loss.

2008 - News - Attacking Alzheimer's with Red Wine and Marijuana.

2008 - News - Marijuana reduces memory impairment.

2008 - News ~ Scientists are High on Idea that Cannabis Reduces Memory Impairment.

2008 - News ~ Israeli Research Shows Cannabidiol May Slow Alzheimer's Disease.

2008 - News ~ Marijuana may be good for the aging brain.

2008 - News ~ Alzheimer's sufferers may benefit from cannabis compound.

2008 - News ~ Cannabis 'could stop dementia in its tracks'.

2008 - News ~ LSUHSC research reports new method to protect brain cells from diseases like Alzheimer's.

2008 - News ~ Could Marijuana Substance Help Prevent Or Delay Memory Impairment In The Aging Brain?

2008 - News ~ Attacking Alzheimer's with Red Wine and Marijuana.

2008 - News ~ Can cannabis offer hope for Alzheimer's?

2008 - News ~ Cannabis-derived medicines may help Alzheimer's.

2009 - Study - The activation of cannabinoid CB2 receptors stimulates in situ and in vitro beta-amyloid removal by human macrophages.

2009 - Study ~ Emerging Role of the CB2 Cannabinoid Receptor in Immune Regulation and Therapeutic Prospects.

2009 - Study ~ Cannabidiol: a promising drug for neurodegenerative disorders?

2009 - Study ~ Endocannabinoids prevent lysosomal membrane destabilisation evoked by treatment with β-amyloid in cultured rat cortical neurons.

2009 - News ~ Medical Marijuana and Alzheimer's Disease.

2010 - Study ~ Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: a neuroprotective therapeutic modality.

2010 - Study ~ Cannabinoid agonist WIN-55,212-2 partially restores neurogenesis in the aged rat brain.

2010 - Study ~ Cannabinoids and Dementia: A Review of Clinical and Preclinical Data.

2010 - Study ~ The development of cannabinoid CBII receptor agonists for the treatment of central neuropathies.

2010 - Study ~ Endocannabinoids Prevent β-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons.

2010 - Study ~ The endocannabinoid system in gp120-mediated insults and HIV-associated dementia.

2010 - Study ~ The Multiplicity of Action of Cannabinoids: Implications for Treating Neurodegeneration.

2010 - News ~ Marijuana could prevent Alzheimer's.

2010 - News ~ Newly discovered mechanism controls levels and efficacy of a marijuana-like substance in the brain.

2011 - Patent ~ US Patent Application 20110257256 - CANNABINOIDS FOR USE IN TREATING OR PREVENTING COGNITIVE IMPAIRMENT AND DEMENTIA.

2011 - Study ~ Cannabidiol and other cannabinoids reduce microglial activation in vitro and in vivo: relevance to Alzheimers' disease.

2011 - Study ~ Cannabidiol Reduces Aβ-Induced Neuroinflammation and Promotes Hippocampal Neurogenesis through PPARγ Involvement.

2011 - Study ~ Gadolinium-HU-308-incorporated micelles.

2011 - Study ~ Anandamide and its congeners inhibit human plasma butyrylcholinesterase. Possible new roles for these endocannabinoids?

2011 - Study ~ Cannabidiol as an emergent therapeutic strategy for lessening the impact of inflammation on oxidative stress.

2011- Study ~ An amyloid β(42)-dependent deficit in anandamide mobilization is associated with cognitive dysfunction in Alzheimer's disease.

2011 - Study ~ Molecular reorganization of endocannabinoid signalling in Alzheimer's disease.

2011 - Study ~ Palmitoylethanolamide counteracts reactive astrogliosis induced by beta-amyloid peptide.

2011 - Study ~ The role of phytochemicals in the treatment and prevention of dementia.

2011 - Study ~ Early onset of aging-like changes is restricted to cognitive abilities and skin structure in Cnr1(-/-) mice.

2011 - Study ~ Intact cannabinoid CB1 receptors in the Alzheimer's disease cortex.

2011- Study ~ The effects of hempseed meal intake and linoleic acid on Drosophila models of neurodegenerative diseases and hypercholesterolemia.

2011 - Study ~ Endocannabinoid 2-arachidonoylglycerol protects neurons against β-amyloid insults.

2011 - News ~ New metabolic pathway for controlling brain inflammation.

2012 - Study ~ Prolonged oral Cannabinoid Administration prevents Neuroinflammation, lowers beta-amyloid Levels and improves Cognitive Performance in Tg APP 2576 Mice.

2012 - Study ~ Can the benefits of cannabinoid receptor stimulation on neuroinflammation, neurogenesis and memory during normal aging be useful in AD prevention?

2012 - Study ~ Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-α.

2012 - Study ~ Activation of the CB(2) receptor system reverses amyloid-induced memory deficiency.

2012 - Study ~ Protective effect of cannabinoid CB1 receptor activation against altered intrinsic repetitive firing properties induced by Aβ neurotoxicity.

2012 - Study ~ Contrasting protective effects of cannabinoids against oxidative stress and amyloid-β evoked neurotoxicity in vitro.

2012 - Study ~ [(125)I]SD-7015 reveals fine modalities of CB(1) cannabinoid receptor density in the prefrontal cortex during progression of Alzheimer's disease.

2012 - Study ~ CB1 Agonist ACEA Protects Neurons and Reduces the Cognitive Impairment of AβPP/PS1 Mice.

2012 - Study ~ CB1 cannabinoid receptor activation rescues amyloid β-induced alterations in behaviour and intrinsic electrophysiological properties of rat hippocampal CA1 pyramidal neurones.

2012 - Study ~ The therapeutic potential of the endocannabinoid system for Alzheimer's disease.

2012 - Study ~ WIN55212-2 attenuates amyloid-beta-induced neuroinflammation in rats through activation of cannabinoid receptors and PPAR-γ pathway.

2012- Study ~ CB(2) receptor and amyloid pathology in frontal cortex of Alzheimer's disease patients.

2012 - Study ~ In vivo type 1 cannabinoid receptor availability in Alzheimer’s disease.

2012 - Book Excerpt ~ How Weed Can Protect Us From Cancer and Alzheimer's.

2012- News ~ Marijuana Compound Found Superior To Drugs For Alzheimer's

2012- News ~ How Cannabinoids May Slow Brain Aging

2012- News ~ 5 Marijuana Compounds That Could Help Combat Cancer, Alzheimers, Parkinsons (If
Only They Were Legal)

2012- News ~ Cannabinoid Receptor Stimulator Reverses Symptoms of Alzheimer's Disease in Animal
Model

2012- News ~ Researchers investigating potential drug for treatement of Alzheimer's disease

2012- News ~ LSUHSC research identifies new therapeutic target for Alzheimer's disease

2012- News ~ Natural Cannabinoids Improve Dopamine Neurotransmission and Tau and Amyloid
Pathology in a Mouse Model of Tauopathy.

2013- Study ~ CB2 Receptor Deficiency Increases Amyloid Pathology and Alters Tau Processing in a
Transgenic Mouse Model of Alzheimer's Disease.

2013- Study ~ Neuroglial Roots of Neurodegenerative Diseases: Therapeutic Potential of
Palmitoylethanolamide in Models of Alzheimer’s Disease

2013- Study ~ Effects of magnolol on impairment of learning and memory abilities induced by
scopolamine in mice
.

2013- Study ~ Activation of the CB(2) receptor system reverses amyloid-induced memory deficiency

2013- Study ~ CB(2) receptor and amyloid pathology in frontal cortex of Alzheimer's disease patients

2013- Study ~ CB2 Cannabinoid Receptor Agonist Ameliorates Alzheimer-Like Phenotype in AβPP/PS1 Mice.

2013- Study ~ Multitarget Cannabinoids as Novel Strategy for Alzheimer Disease.

2013- Study ~ Implication of JNK pathway on tau pathology and cognitive decline in a senescence-
accelerated mouse model.

 
2013- Study ~ Cannabinoid receptor 1 deficiency in a mouse model of Alzheimer's disease leads to
enhanced cognitive impairment despite of a reduction in amyloid deposition

2013- Study ~ Therapeutic Potential of Cannabinoids in Neurodegenerative Disorders: A Selective
Review.

2013- Study ~ Cannabinoid Effects on β Amyloid Fibril and Aggregate Formation, Neuronal and
Microglial-Activated Neurotoxicity In Vitro

2013- Study ~ The Influence of Cannabinoids on Generic Traits of Neurodegeneration.

2013- Study ~ In vivo type 1 cannabinoid receptor availability in Alzheimer's disease.

2013- Study ~ Cannabidiol Normalizes Capase 3, Synatophsin, and Mitochondrial Fission Protein
DNM1L Expression Levels in Rats with Brain Iron Overload: Implications for Neuroprotection

2013- Study ~ Cannabidiol Promotes Amyloid Precursor Protein Ubiquitination and Reduction of Beta
Amyloid Expression in SHSY5YAPP+ Cells Through PPARγ Involvement.

2013- Study ~ Cannabinoid agonists showing BuChE inhibition as potential therapeutic agents for
Alzheimer's disease.

 
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MARIJUANA SLOWS ALZHEIMER'S DECLINE

Pubdate: Thu, 24 Feb 2005
Source: Jerusalem Post (Israel)
Copyright: 2005 The Jerusalem Post
Contact: http://info.jpost.com/C002/Services/Feedback/editors.html
Website: http://www.jpost.com/
Details: http://www.mapinc.org/media/516
Author: Judy Siegel-Itzkovich
Bookmark: http://www.mapinc.org/mmj.htm (Cannabis - Medicinal)

STUDY: MARIJUANA SLOWS ALZHEIMER'S DECLINE

New Spanish and Israeli research shows that a synthetic analogue of the active component of marijuana can reduce the inflammation and prevent the mental decline associated with Alzheimer's disease.  Although it was conducted on human brain tissue in the lab and in a rat model -- but not in living humans -- the research is regarded as a major step not only in understanding how the brain reacts to Alzheimer's disease, but also in helping to develop novel drugs for Alzheimer's and even Parkinson's disease.

Prof.  Raphael Mechoulam, a medicinal chemistry expert who discovered marijuana's active component ( called THC ), conducted the study with researchers at the Cajal Institute and Complutense University in Madrid, led by Maria de Ceballos.  The study appears in Wednesday's issue of The Journal of Neuroscience, which is published by the Society for Neuroscience, an organization of more than 36,000 basic scientists and clinicians who study the brain and nervous system.

To show the preventive effects of cannabinoids on Alzheimer's disease, the team first compared the brain tissue of patients who died from Alzheimer's disease with that of healthy people who had died at a similar age.  They looked closely at cannabinoid receptors CB1 and CB2 - proteins to which cannabinoids bind, allowing their effects to be felt - and atmicroglia, which activate the brain s immune response.  Micro-glia collect near plaques and, when active, cause inflammation.  The researchers found a dramatically reduced functioning of cannabinoid receptors in diseased brain tissue, meaning that patients had lost the capacity to experience cannabinoids' protective effects.

In addition, the researchers showed that cannabinoids prevented cognitive decline through rat experiments.  They injected either amyloid ( which leads to cognitive decline ) that had been allowed to aggregate or control proteins into the brains of rats for one week.  Other rats were injected with a cannabinoid and either amyloid or a control protein.  After two months, the researchers trained the rats over five days to find a platform hidden underwater.  Rats treated with the control protein - with or without cannabinoids - and those treated with the amyloid protein and cannabinoid were able to find the platform.  Rats treated with amyloid protein alone did not learn how to find the platform.

Meshoullam said that the discovery was important, since most drugs given for neurodegenerative diseases like Alzheimer's and Parkinson's are work merely against symptoms and not the cause and essence of the neurodegeneration.  It is not necessary to smoke marijuana to conduct trials, but to use the synthetic versions of the active ingredient, he told The Jerusalem Post.

Clinical trials have not yet been scheduled or a request made for approval.  It is very complicated and expensive to run clinical trials, he said, but he hoped they would be carried out due to the massive threat to human health of Alzheimer's and other neurodegenerative disorders.

The researchers found that the presence of amyloid protein in the rats' brains activated immune cells.  Rats that received the control protein alone or cannabinoid and a control protein did not show activation of microglia.  Using cell cultures, the investigators confirmed that cannabinoids counteracted the activation of microglia and thus reduced inflammation. 

These findings that cannabinoids work both to prevent inflammation and to protect the brain may set the stage for their use as a therapeutic approach for Alzheimer's disease, de Ceballos said.  The scientists will now focus their efforts on targeting one of the two main cannabinoid receptors that is not involved in producing the psychotropic effects, or high, from marijuana.

 

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Marijuana may block Alzheimer's

Brain
The compound may protect the brain
The active ingredient in marijuana may stall decline from Alzheimer's disease, research suggests.

Scientists showed a synthetic version of the compound may reduce inflammation associated with Alzheimer's and thus help to prevent mental decline.

They hope the cannabinoid may be used to developed new drug therapies.

The research, by Madrid's Complutense University and the Cajal Institute, is published in the Journal of Neuroscience.

We would warn the public against taking marijuana as a way of preventing Alzheimer's
The scientists first compared the brain tissue of patients who died from Alzheimer's disease with that of healthy people who had died at a similar age.

 

They looked closely at brain cell receptors to which cannabinoids bind, allowing their effects to be felt.

They also studied structures called microglia, which activate the brain's immune response.

Microglia collect near the plaque deposits associated with Alzheimer's disease and, when active, cause inflammation.

The researchers found a dramatically reduced functioning of cannabinoid receptors in diseased brain tissue.

This was an indication that patients had lost the capacity to experience cannabinoids' protective effects.

The next step was to test the effect of cannabinoids on rats injected with the amyloid protein that forms Alzheimer's plaques.

Those animals who were also given a dose of a cannabinoid performed much better in tests of their mental functioning.

The researchers found that the presence of amyloid protein in the rats' brains activated immune cells.

However, rats that also received the cannabinoid showed no sign of microglia activation.

Using cell cultures, the researchers confirmed that cannabinoids counteracted the activation of microglia and thus reduced inflammation.

Drug target

Researcher Dr Maria de Ceballos said: "These findings that cannabinoids work both to prevent inflammation and to protect the brain may set the stage for their use as a therapeutic approach for Alzheimer's disease."

Dr Susanne Sorensen, head of research at the Alzheimer's Society, said: "This is important research because it provides another piece of the jigsaw puzzle on the workings of the brain.

"There is no cure for Alzheimer's disease, so the identification of another target for drug development is extremely welcome.

"The Alzheimer's Society looks forward to seeing further research being carried out on cannabinoid receptors as drug targets for Alzheimer's disease but would warn the public against taking marijuana as a way of preventing Alzheimer's.

"It is now generally recognised that as well as providing a 'high', long-term use of marijuana can also lead to depression in many individuals."

Different receptors

Harriet Millward, of the Alzheimer's Research Trust, said there were two main types of cannabinoid receptor, CR1 and CR2.

"It is CR1 that produces most of the effects of marijuana, including the harmful ones.

"If it is possible to make drugs that act only on CR2, as suggested by the authors of this study, they might mimic the positive effects of cannabinoids without the damaging ones of marijuana.

"However, this is a fairly new field of research and producing such selective drugs is not an easy task.

"There is also no evidence yet that cannabinoid-based drugs can slow the decline in human Alzheimer's patients."

 

 

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Prevention of Alzheimer's Disease Pathology by Cannabinoids

Neurobiology of Disease

Neuroprotection Mediated by Blockade of Microglial Activation

Belén G. Ramírez, Cristina Blázquez, Teresa Gómez del Pulgar, Manuel Guzmán, and María L. de Ceballos

Neurodegeneration Group, Cajal Institute, Consejo Superior de Investigaciones Científicas, 28002 Madrid, Spain, and Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain

Alzheimer's disease (AD) is characterized by enhanced {beta}-amyloid peptide ({beta}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.

This background prompted us to study the localization, expression, and function of cannabinoid receptors in AD and the possible protective role of cannabinoids after {beta}A 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 {beta}A-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block {beta}A-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 {beta}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.



Received Sep 9, 2004; revised December 28, 2004; accepted December 30, 2004.

Articles citing this article

Marijuana's Active Ingredient Shown to Inhibit Primary Marker of Alzheimer's Disease

Discovery Could Lead to More Effective Treatments

LA JOLLA, CA, August 9, 2006 - Scientists at The Scripps Research Institute have found that the active ingredient in marijuana, tetrahydrocannabinol or THC, inhibits the formation of amyloid plaque, the primary pathological marker for Alzheimer's disease. In fact, the study said, THC is "a considerably superior inhibitor of [amyloid plaque] aggregation" to several currently approved drugs for treating the disease.

The study was published online August 9 in the journal Molecular Pharmaceutics, a publication of the American Chemical Society.

According to the new Scripps Research study, which used both computer modeling and biochemical assays, THC inhibits the enzyme acetylcholinesterase (AChE), which acts as a "molecular chaperone" to accelerate the formation of amyloid plaque in the brains of Alzheimer victims. Although experts disagree on whether the presence of beta-amyloid plaques in those areas critical to memory and cognition is a symptom or cause, it remains a significant hallmark of the disease. With its strong inhibitory abilities, the study said, THC "may provide an improved therapeutic for Alzheimer's disease" that would treat "both the symptoms and progression" of the disease.

"While we are certainly not advocating the use of illegal drugs, these findings offer convincing evidence that THC possesses remarkable inhibitory qualities, especially when compared to AChE inhibitors currently available to patients," said Kim Janda, Ph.D., who is Ely R. Callaway, Jr. Professor of Chemistry at Scripps Research, a member of The Skaggs Institute for Chemical Biology, and director of the Worm Institute of Research and Medicine. "In a test against propidium, one of the most effective inhibitors reported to date, THC blocked AChE-induced aggregation completely, while the propidium did not. Although our study is far from final, it does show that there is a previously unrecognized molecular mechanism through which THC may directly affect the progression of Alzheimer's disease."

As the new study points out, any new treatment that could halt or even slow the progression of Alzheimer's disease would have a major impact on the quality of life for patients, as well as reducing the staggering health care costs associated with the disease.

 

Alzheimer's disease is the leading cause of dementia among the elderly, and the numbers are growing. The Alzheimer's Association estimates 4.5 million Americans have the disease, a figure that could reach as high as 16 million by 2050. A survey by the National Center for Health Statistics noted that half of all nursing home residents have Alzheimer's disease or a related disorder. The costs of caring for Alzheimer's patients are at least $100 billion annually, according to the National Institute on Aging.

Over the last two decades, the causes of Alzheimer's disease have been clarified through extensive biochemical and neurobiological studies, leading to an assortment of possible therapeutic strategies including interference with beta amyloid metabolism, the focus of the Scripps Research study.

The cholinergic system - the nerve cell system in the brain that uses acetylcholine (Ach) as a neurotransmitter - is the most dramatic of the neurotransmitter systems affected by Alzheimer's disease. Levels of acetylcholine, which was first identified in 1914, are abnormally low in the brains of Alzheimer's patients. Currently, there are four FDA-approved drugs that treat the symptoms of Alzheimer's disease by inhibiting the active site of acetylcholinesterase, the enzyme responsible for the degradation of acetylcholine.

"When we investigated the power of THC to inhibit the aggregation of beta-amyloid," Janda said, "we found that THC was a very effective inhibitor of acetylcholinesterase. In addition to propidium, we also found that THC was considerably more effective than two of the approved drugs for Alzheimer's disease treatment, donepezil (Aricept ®) and tacrine (Cognex ®), which reduced amyloid aggregation by only 22 percent and 7 percent, respectively, at twice the concentration used in our studies. Our results are conclusive enough to warrant further investigation."

Other authors of the study, titled "A Molecular Link Between the Active Component of Marijuana and Alzheimer's Disease Pathology," include Lisa M. Eubanks, Claude J. Rogers, and Tobin J. Dickerson of The Scripps Research Institute, the Skaggs Institute for Chemical Biology, and the Worm Institute for Research and Medicine; and Albert E. Beuscher IV, George F. Koob, and Arthur J. Olson of The Scripps Research Institute.

The study was supported by the Skaggs Institute for Chemical Biology at Scripps Research and the National Institutes of Health.

About The Scripps Research Institute

The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations, at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel. Scripps Research is headquartered in La Jolla, California. It also includes Scripps Florida, whose researchers focus on basic biomedical science, drug discovery, and technology development. Currently operating from temporary facilities in Jupiter, Scripps Florida will move to its permanent campus in 2009.

For more information contact:
Office of Communications
10550 North Torrey Pines Road
La Jolla, California 92037
[email protected]

 

 

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Safety and efficacy of Dronabinol in the treatment of agitation in patients with Alzheimer's disease with anorexia

TitleSafety and efficacy of dronabinol in the treatment of agitation in patients with Alzheimer’s disease with anorexia: A retrospective chart review
Author(s)Patel S, Shua-Haim JR, Pass M
Journal, Volume, IssueAbstract, International Psychogeriatric Association, Eleventh International Congress, 2003
Major outcome(s)Weight gain in all, reduction of agitation in 65%.
 
IndicationAppetite loss/weight loss;Alzheimer's diseaseAbstract
MedicationDelta-9-THC

Objective: to investigate the safety and efficacy of dronabinol in Alzheimer’s disease (AD) patients with agitation.
Design: retrospective review.
Materials and Methods: AD patients with anorexia, where according to family members or caregivers (staff nurses) agitation was not satisfactorily controlled, were prescribed dronabinol. All met the DSM-IV & NINCDS-ADRDA criteria for possible AD. There were no exclusion criteria. Study subjects resided in a dementia unit in assistant living facility and in a nursing home. Dronabinol, 5 mg/day in 2 divided doses was given initially & titrated up to a maximum of 10 mg/day. Cognition and function evaluated at the start and end of the study period. MMSE and ADL scales used for assessment. Concomitant medications were recorded. After 1 month of treatment, caregivers were asked to complete a questionnaire regarding their impression on treatment efficacy.
Results: 48 patients were treated with dronabinol. Average age was 77. Average MMSE was 16. All patients were treated with atypical neuroleptics. All were treated with multiple (greater than 3) medications to control behavior. All patients had diagnoses of anorexia prior to initiation of dronabinol therapy. Weight gain was reported in all patients. Agitation significantly improved in 31 (65%). In 14 (37%) there was improvement in MMSE score (average increase by 1.6 points (range +1 to +3). Functional improvement was reported in 33 (69%). 17 (35%) patients had no significant beneficial effect with regard to agitation, and their medication was discontinued within 2 weeks of initiation of treatment. No patient experienced any significant adverse event (falls, syncope, seizures or exacerbation of agitation/depression).
Conclusion: Dronabinol treatment for agitation in AD patients with anorexia was effective in 31 out of 48 of patients who were refractory to other medications. No adverse events were reported.

Route(s)Oral
Dose(s) 
Duration (days)30
Participants48 Alzheimer patients
DesignOpen study
Type of publicationMeeting abstract
Address of author(s)Meridian Institute for Aging, Manchester Township, NJ, USA
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Open-label study of Dronabinol in the treatment of refractory agitation in Alzheimer's disease

TitleOpen-label study of dronabinol in the treatment of refractory agitation in Alzheimer’s disease: a pilot study
Author(s)Ross JS, Shua-Haim JR
Journal, Volume, IssueAbstract, American Society of Consultant Pharmacists' 34th Annual Meeting, November 12-15, 2003.
Major outcome(s)Significant reduction of agitation
 
IndicationAlzheimer's diseaseAbstract
MedicationDelta-9-THC

Objectives: Primary, to investigate the efficacy of two doses of dronabinol for the treatment of behavioral agitation in community-dwelling patients with Alzheimer’s disease (AD). Secondary, to evaluate two doses of dronabinol in improving the patient’s global functioning and to determine the effects of two doses of dronabinol on the caregiver’s burden, strain (distress), and quality of life.

Design: A phase II, open-label, eight-week study in a total of 54 patients with AD. Twenty-seven patients were randomly assigned to Group 1—dronabinol 2.5 mg bid and 27 to Group 2—dronabinol 5.0 mg bid. The primary efficacy measurement was the Cohen-Mansfield Agitation Inventory (CMAI), a 38-item rating scale that evaluates the prevalence of pathological and disruptive behaviors, rating each on a seven-point scale of frequency ranging from 0 to 6. The secondary efficacy measurements were the Caregiver’s Burden Inventory (CBI), CGI Severity of Alzheimer’s Disease (CGI-S AD), Instrumental Activities of Daily Living scale (IADL), and Mini-Mental State Examination (MMSE).

Results: Significant reductions in CMAI scores were observed at both dronabinol dose levels (2.5: P<0.001, 5.0: P=0.024). The difference between the two groups was not statistically significant. Percent reductions in CMAI scores were statistically significant for both groups (2.5: P<0.001, 5.0: P=0.048). There was a trend toward a decrease in CBI scores, with no statistical difference between treatment groups. There was a trend toward a decrease in CGI-S AD scores in the dronabinol 5.0-mg bid group. There was a trend toward an increase in IADL scores with no difference between groups. There was no difference between groups in MMSE.
Conclusion: The results from the CMAI assessment indicated that dronabinol at both 2.5 mg bid and 5.0 mg bid were effective treatments for behavioral agitation in community-dwelling AD patients. There was not a significant difference between the doses of dronabinol in CMAI scores and most secondary efficacy parameters.

Benefit: Dronabinol was found to be an effective treatment for behavioral agitation in community-dwelling patients with AD.

Route(s)Oral
Dose(s)2 x 2,5 mg or 2 x 5 mg
Duration (days)56
Participants54 patients with AD
DesignOpen study
Type of publicationMeeting abstract
Address of author(s)Monmouth Medical Center, Long Branch, NJ, USA
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Effects of dronabinol on anorexia and disturbed behavior in patients with Alzheimer's disease.

TitleEffects of dronabinol on anorexia and disturbed behavior in patients with Alzheimer's disease.
Author(s)Volicer L, Stelly M, Morris J, McLaughlin J, Volicer BJ
Journal, Volume, IssueInternational Journal of Geriatric Psychiatry 1997;12(9):913-919
Major outcome(s)higher weight gain with THC; reduction of disturbed behaviour with THC
 
IndicationAppetite loss/weight loss;Alzheimer's diseaseAbstract
MedicationDelta-9-THC

A placebo-controlled crossover design, with each treatment period lasting 6 weeks, was used to investigate effects of dronabinol in 15 patients with a diagnosis of probable Alzheimer's disease who were refusing food. Eleven patients completed both study periods; one patient who died of a heart attack 2 weeks before the end of the study was also included in the analysis. The study was terminated in 3 patients: one developed a grand mal seizure and 2 developed serious intercurrent infections. Body weight of study subjects increased more during the dronabinol treatment than during the placebo periods. Dronabinol treatment decreased severity of disturbed behavior and this effect persisted during the placebo period in patients who received dronabinol first. Adverse reactions observed more commonly during the dronabinol treatment than during placebo periods included euphoria, somnolence and tiredness, but did not require discontinuation of therapy. These results indicate that dronabinol is a promising novel therapeutic agent which may be useful not only for treatment of anorexia but also to improve disturbed behavior in patients with Alzheimer's disease.

Cannabinoids reduce the progression of Alzheimer's disease in animals

                       IACM-Bulletin of 06 March 2005

Science: Cannabinoids reduce the progression of Alzheimer's disease in animals

Research by scientists of Madrid's Complutense University and the Cajal Institute published in the Journal of Neuroscience has demonstrated that cannabinoids can reduce pathological processes associated with Alzheimer's disease. Researchers hope that cannabinoids may be used to develop new drug therapies against the disease.

They first compared the brain tissue of patients who died from Alzheimer's disease with that of healthy people who had died at a similar age. The researchers found a dramatically reduced functioning of cannabinoid receptors in diseased brain tissue and markers of microglia activation. Microglia activate the brain's immune response and are found near the plaque deposits associated with Alzheimer's disease. When active, microglia cause inflammation. Nerve cells with cannabinoid-1 receptors (CB1), present in high numbers in control subjects, were greatly reduced in areas of microglial activation.

In a second step rats were injected with amyloid-beta peptide. This protein plays an important role in Alzheimer's disease, since increased brain levels of amyloid-beta are supposed to result in aggregation of this protein to form plaques. Animals who also received different cannabinoids performed better in tests of their mental functioning. Analyses showed that cannabinoids had prevented microglial activation and thus had reduced inflammation. These effects were also mediated by cannabinoids that only bind to CB2 receptors.

Researchers concluded: "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."

British researchers, who published their work in the journal Sub-Cellular Biochemistry, found that phosphorylation of amyloid-beta increased the neurotoxicity of this protein. And they demonstrated that cannabinoids prevented these damaging effects of phosphorylated amyloid-beta on nerve cells.

(Sources: Ramirez BG, et al. Prevention of Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation. J Neurosci 2005;25(8):1904-13; Milton NG. Phosphorylated amyloid-beta: the toxic intermediate in alzheimer's disease neurodegeneration. Subcell Biochem 2005;38:381-402; BBC News of 22 February 2005)

UK/USA: GW Pharmaceuticals accelerates plans to introduce Sativex in the USA

The British company GW Pharmaceuticals said on 28 February it was accelerating plans to introduce its cannabis-based medicines into the United States. GW said it had engaged the U.S.-based Apjohn Group, a 10-member group of former major U.S. pharmaceutical company executives with extensive experience in clinical development, regulatory affairs and public policy.

GW's Sativex has won qualified approval in Canada for the treatment of neuropathic pain in multiple sclerosis in December 2004, but approval in Great Britain for the treatment of spasticity in multiple sclerosis has been repeatedly delayed. Sativex is an under-the-tongue cannabis spray containing equal amounts of THC and CBD.

(Source: Reuters of 28 February 2005)

News in brief

Canada: Tax relief for medical cannabis
Canadians will get tax relief to buy medical cannabis under the federal budget proposed by finance minister Ralph Goodale. Marijuana bought for medical purposes from Health Canada or a designated grower will be eligible for tax relief after the annual medical expenses exceed 3 per cent of net income or 1,844 Canadian dollars. (Source: Toronto Star of 24 February 2005)

 

Austria: New research centre
A new research centre for plant derived drugs will be founded in Innsbruck. The main focuses of Bionorica Research will be the investigation of plant derived airway therapeutics and the investigation of cannabis products for medical purposes. A subsidiary company of Bionorica in Germany, Delta 9 Pharma, already produces the cannabis compound dronabinol (THC) for medical uses. (Source: Der Standard of 28 February 2005)

USA: New Mexico
The state Senate of New Mexico has approved three separate bills that would allow the medical use of cannabis, but it is unclear whether the House of Representatives will support them. Under one of the measures, the cannabis would be grown at licensed, secure facilities and then distributed to patients who were registered to possess and smoke it. An alternative bill requires the medical marijuana to be manufactured by a drug company. The third bill the Senate endorsed would allow the use of marijuana only topically, such as in an ointment. (Source: Associated Press of 2 March 2005)

Australia: Australian Capital Territory
Changes of the drug laws of the Australian Capital Territory (ACT), one of the Australian states, came into effect on 6 March. They reduce the number of cannabis plants that can be cultivated without being charged with a criminal offence from five to two plants. The criminal law allows a person to posses up to 25 grams of dried cannabis for personal use. (Source: Government of ACT of 1 March 2005, www.cmd.act.gov.au)

 
 

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Molecular Link between the Active Component of Marijuana and Alzheimer's Disease Pathology

A molecular link between the active component of marijuana and Alzheimer's disease pathology. [Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
Mol Pharm 2006 Nov-Dec; 3(6):773-7.

Alzheimer's disease is the leading cause of dementia among the elderly, and with the ever-increasing size of this population, cases of Alzheimer's disease are expected to triple over the next 50 years. Consequently, the development of treatments that slow or halt the disease progression have become imperative to both improve the quality of life for patients and reduce the health care costs attributable to Alzheimer's disease. Here, we demonstrate that the active component of marijuana, Delta9-tetrahydrocannabinol (THC), competitively inhibits the enzyme acetylcholinesterase (AChE) as well as prevents AChE-induced amyloid beta-peptide (Abeta) aggregation, the key pathological marker of Alzheimer's disease. Computational modeling of the THC-AChE interaction revealed that THC binds in the peripheral anionic site of AChE, the critical region involved in amyloidgenesis. Compared to currently approved drugs prescribed for the treatment of Alzheimer's disease, THC is a considerably superior inhibitor of Abeta aggregation, and this study provides a previously unrecognized molecular mechanism through which cannabinoid molecules may directly impact the progression of this debilitating disease.

 

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THC inhibits primary marker of Alzheimer's disease

Scientists at The Scripps Research Institute in La Jolla, California, have found that THC inhibits the formation of amyloid plaque, the primary pathological marker for Alzheimer's disease. The study to be published in Molecular Pharmaceutics says, THC is "a considerably superior inhibitor of [amyloid plaque] aggregation" to several currently approved drugs for treating the disease.

According to the new experimental study THC inhibits a protein, which acts as a accelerator of the formation of amyloid plaque in the brains of Alzheimer victims. Although experts disagree on whether the presence of beta-amyloid plaques in those areas critical to memory and cognition is a symptom or cause, it remains a significant hallmark of the disease. With its strong inhibitory abilities, the study said, THC "may provide an improved therapeutic for Alzheimer's disease" that would treat "both the symptoms and progression" of the disease.

(Source: Press release by the Scripps Research Institute of 9 August 2006, www.scripps.edu/news/press/080906.html)

 

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Cannabinoid receptor stimulation is anti-inflammatory and improves memory in old rats

Neurobiol Aging. 2008 Dec;29(12):1894-901. Epub 2007 Jun 11.

Marchalant Y, Cerbai F, Brothers HM, Wenk GL.

Department of Psychology, Psychology Building, Ohio State University, Columbus, OH 43210, USA. [email protected]

Abstract

The number of activated microglia increase during normal aging. Stimulation of endocannabinoid receptors can reduce the number of activated microglia, particularly in the hippocampus, of young rats infused chronically with lipopolysaccharide (LPS). In the current study we demonstrate that endocannabinoid receptor stimulation by administration of WIN-55212-2 (2mg/kg day) can reduce the number of activated microglia in hippocampus of aged rats and attenuate the spatial memory impairment in the water pool task. Our results suggest that the action of WIN-55212-2 does not depend upon a direct effect upon microglia or astrocytes but is dependent upon stimulation of neuronal cannabinoid receptors. Aging significantly reduced cannabinoid type 1 receptor binding but had no effect on cannabinoid receptor protein levels. Stimulation of cannabinoid receptors may provide clinical benefits in age-related diseases that are associated with brain inflammation, such as Alzheimer's disease.

PMID: 17561311 [PubMed - indexed for MEDLINE]PMCID: PMC2586121Free PMC Article

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Alzheimer's disease; taking the edge off with cannabinoids?

V A Campbell1* and A Gowran1
1Department of Physiology and Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland
Received May 29, 2007; Revised July 3, 2007; Accepted August 8, 2007.
Abstract
 
Alzheimer's disease is an age-related neurodegenerative condition associated with cognitive decline. The pathological hallmarks of the disease are the deposition of β-amyloid protein and hyperphosphorylation of tau, which evoke neuronal cell death and impair inter-neuronal communication.
 
The disease is also associated with neuroinflammation, excitotoxicity and oxidative stress. In recent years the proclivity of cannabinoids to exert a neuroprotective influence has received substantial interest as a means to mitigate the symptoms of neurodegenerative conditions. In brains obtained from Alzheimer's patients alterations in components of the cannabinoid system have been reported, suggesting that the cannabinoid system either contributes to, or is altered by, the pathophysiology of the disease. Certain cannabinoids can protect neurons from the deleterious effects of β-amyloid and are capable of reducing tau phosphorylation. The propensity of cannabinoids to reduce β-amyloid-evoked oxidative stress and neurodegeneration, whilst stimulating neurotrophin expression neurogenesis, are interesting properties that may be beneficial in the treatment of Alzheimer's disease. Δ9-tetrahydrocannabinol can also inhibit acetylcholinesterase activity and limit amyloidogenesis which may improve cholinergic transmission and delay disease progression.
 
Targeting cannabinoid receptors on microglia may reduce the neuroinflammation that is a feature of Alzheimer's disease, without causing psychoactive effects. Thus, cannabinoids offer a multi-faceted approach for the treatment of Alzheimer's disease by providing neuroprotection and reducing neuroinflammation, whilst simultaneously supporting the brain's intrinsic repair mechanisms by augmenting neurotrophin expression and enhancing neurogenesis. The evidence supporting a potential role for the cannabinoid system as a therapeutic target for the treatment of Alzheimer's disease will be reviewed herewith.


Pathophysiology of Alzheimer's disease
Alzheimer's disease (AD) is a chronic debilitating neurodegenerative condition that is associated with progressive cognitive decline and profound neuronal loss, and estimated to affect 10% of people over the age of 65 years and 25% of people over the age of 80 years (Herbert et al., 2003). Western society is developing an increasingly aged population and this demographic shift is associated with a rise in the prevalence of age-related illnesses such as AD.
 
The United Nations population projections estimate that 370 million people will be older than 80 years by 2050 and the associated increase in patients with AD will pose a substantial socio-economic burden. While a small proportion of AD cases have a genetic basis, the majority of cases are sporadic with unknown aetiology. A consistent feature of the AD brain is the presence of senile plaques composed of pathogenic extracellular deposits of β-amyloid (Aβ), a 1–42 amino acid peptide derived from aberrant processing of the transmembrane amyloid precursor protein (Walsh and Selkoe, 2007). Aβ fragments are proposed to play a central role in the genesis of the disease by evoking neuronal cell death (Boland and Campbell, 2003).
 
The senile plaques are located within various brain regions but the hippocampus, cerebral cortex and amygdala are particularly vulnerable and plaques begin to form in these regions early in the disease process resulting in memory loss and behavioural changes (Ogomori et al., 1989). A second pathological hallmark of the disease is the hyperphosphorylation of the microtubule-associated protein, tau, resulting in formation of the intracellular neurofibrillary tangles that impair inter-neuronal communication (Mi and Johnson, 2006).
 
AD is also associated with neuroinflammatory events and oxidative stress that are likely to exacerbate the disease process. Epidemiological studies support an involvement of inflammatory mechanisms in AD since patients using non-steroidal anti-inflammatory drugs for a 2-year period have a 60–80% reduction in the risk for the disease, while long-term non-steroidal anti-inflammatory drug treatment attenuates disease onset and reduces the severity of symptoms (Rich et al., 1995). Microglia are the Principal immune cells in the brain and in the AD brain they surround the senile plaques, possibly recruited to the plaque region in an attempt to clear the Aβ burden by phagocytosis (Wilkinson and Landreth, 2006).
 
In AD, the Aβ deposition exceeds the phagocytic ability of the microglia and the persistent presence of activated microglia at the plaque results in a prolonged release of proinflammatory cytokines such as interleukin-1β (Bayer et al., 1999; Heneka and O'Banion, 2007) which mediate local inflammation and have the proclivity to increase the processing of amyloid precursor protein to generate more Aβ fragments (Heneka and O'Banion, 2007), as well as having a direct neurotoxic influence (Vereker et al., 2000).
 
The association of activated microglia at the periphery of the senile plaque contributes to the generation of reactive oxygen species that mediate the oxidative damage found in the brains of patients with AD (Wilkinson and Landreth, 2006). Thus, inflammation and oxidative stress play a critical role in the disease process and anti-inflammatory and antioxidant strategies are likely to have enormous therapeutic potential for AD patients.
 
Other factors that are thought to contribute to the pathophysiology of AD include dysregulation of intracellular calcium homeostasis and excitotoxicity (LaFerla, 2002). Cholinergic neurones are particularly vulnerable in AD and current therapeutics include acetylcholinesterase (AChE) inhibitors that aim to enhance acetylcholine (ACh) availability. However, such drugs are only suitable for the mild cognitive impairment that occurs early in the disease and no treatments are currently available to reverse the progression of the disease.

Cannabinoid system in the brain
The discovery of an endogenous cannabinoid (CB)-signalling system in the brain has prompted much research into understanding how this system regulates physiological and pathological events within the central nervous system. The endocannabinoid molecules, 2-arachidonoyl glycerol and anandamide, interact with the G-protein-coupled cannabinoid receptors, CB1 and CB2. These receptors are also activated by phytocannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), isolated from the Cannabis sativa plant.
 
The action of endocannabinoids at their receptors is terminated by enzymatic degradation of the endocannabinoids, or by membrane transport (Piomelli, 2003). Early reports indicating a potential role for the cannabinoid system in the management of AD are based on the finding that Dronabinol, an oil-based solution of Δ9-THC, improves the disturbed behaviour and stimulates appetite in AD patients (Volicer et al., 1997), and alleviates nocturnal agitation in severely demented patients (Walther et al., 2006).
 
More recently, an increasing body of evidence has accumulated to suggest antioxidant, anti-inflammatory and neuroprotective roles of the cannabinoid system (Jackson et al., 2005). Such properties may be harnessed to circumvent the neurodegenerative process and offer more effective approaches to treat AD (Pazos et al., 2004). In this review the recent experimental evidence that highlights the potential of the cannabinoid system to alleviate some of the pathology and cognitive decline associated with AD will be discussed.

The cannabinoid system in the AD brain
 
The CB1 receptor is abundant within the brain and associated with the cortex, hippocampus, cerebellum and basal ganglia (Herkenham et al., 1991). CB1 receptors in the hippocampus contribute to the effect of cannabinoids on learning and memory (Riedel and Davies, 2005); cognitive processes, which are disrupted early in the course of AD. CB2 receptors have a more limited expression in the central nervous system, being largely confined to neurones within the brainstem (Van Sickle et al., 2005), cerebellum (Ashton et al., 2006) and microglia (Nunez et al., 2004).
 
Post-mortem studies of AD brains have detected increased expression of CB1 and CB2 receptors on microglia within the senile plaque, while CB1 expression is reduced in neurones more remote from the plaque (Ramirez et al., 2005). Also, cannabinoid receptors in the AD brain are nitrosylated, and this may contribute to the impaired coupling of these receptors to downstream effector signalling molecules (Ramirez et al., 2005). Other studies have failed to establish a link between changes in CB1 receptors in the AD brain and the specific pathological events that take place in this illness (Westlake et al. 1994), and report no changes in expression of CB1 receptors in the vicinity of the senile plaque (Benito et al., 2003).
 
However, the endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, is upregulated in the senile plaque (Benito et al., 2003), and may contribute to the increase in expression of anandamide metabolites, such as arachidonic acid, in the vicinity of the senile plaque. Such a pathway may be involved in increasing the production of prostaglandins and related pro-inflammatory molecules that are pertinent to the inflammatory process of AD. The association of fatty acid amide hydrolase with astrocytes within the senile plaque may participate in the astrocytic events that culminate in the reactive gliosis that is observed in regions rich in Aβ deposits (Wyss-Coray, 2006).

Cannabinoids mediate neuroprotection
Neuronal damage can increase the production of endocannabinoids (Stella et al., 1997; Marsicano et al., 2003), and cells lacking CB1 receptors are more vulnerable to damage (Marsicano et al., 2003). Those studies indicate that neural cannabinoid tone influences neuronal survival and suggest that augmentation of the cannabinoid system may offer protection against the deleterious consequences of pathogenic molecules such as Aβ. Recently, Aβ has been demonstrated to induce hippocampal degeneration, gliosis and cognitive decline, with a concomitant increase in the production of the endocannabinoid, 2-arachidonoyl glycerol, and this may reflect an attempt of the endocannabinoid system to provide neuroprotection from Aβ-induced damage (Van Der Stelt et al., 2006). Furthermore, in that study, when endocannabinoid uptake was inhibited by VDM-11, the Aβ-induced neurotoxicity and memory impairment were reversed, although this was dependent upon early administration of the reuptake inhibitor.
 
Those findings suggest that robust and early pharmacological enhancement of brain endocannabinoid levels may protect against the deleterious consequences of Aβ. Other endocannabinoids, such as anandamide and noladin ether, have been found to reduce Aβ neurotoxicity in vitro via activation of the CB1 receptor and engagement the extracellular-regulated kinase pathway (Milton, 2002).
 
Thus, endocannabinoids can reverse the negative consequences of exposure to Aβ, and such findings suggest that drugs designed to augment endocannabinoid tone, including inhibitors of membrane uptake and fatty acid amide hydrolase inhibitors, may have potential in the treatment of AD. However, the study by Van Der Stelt et al. (2006) cautions that the timing of endocannabinoid upregulation by pharmacological intervention in relation to the time-course of development of the disease pathology is crucial, since administration of VDM-11 later in the pathological cascade actually worsens memory retention in rodents.
 
Also, the physiological role of the cannabinoid system in mnemonic processes should not be underestimated. In the hippocampus CB1 receptor activation is negatively associated with the performance of rodents in memory tasks (Castellano et al., 2003), possibly via a reduction in hippocampal ACh levels (Gifford et al., 2000), while the CB1 antagonist, SR141716A improves performance in memory tasks (Wolff and Leander, 2003). Furthermore, the impairment in memory evoked by Aβ in rodents is reversed by SR141716A (Mazzola et al., 2003), suggesting that CB1 receptor blockade may be beneficial in reversing the amnesia associated with AD. However, given the evidence for a neuroprotective role of the CB1 receptor (Marsicano et al., 2003; Alger, 2006), CB1 antagonists pose the risk of exacerbating the neurodegenerative component of the disease, which may negate the beneficial effects of such drugs on amnesia.

Cannabinoids and excitotoxicity
 
The dysregulation of intracellular Ca2+ homeostasis (Smith et al., 2005) and excessive activation of the N-methyl D-aspartate (NMDA) subtype of glutamate receptor, leading to excitotoxicity, are features of the AD brain (Sonkusare et al., 2005).
 
All of the clinical mutations in the presenilin genes (PS1/PS2) that have been linked with the inherited form of AD disrupt calcium signalling (Smith et al., 2005), which may contribute to subsequent neurodegeneration and memory impairments (Rose and Konnerth, 2001).
 
Also, Aβ can itself directly increase voltage-dependent Ca2+ channel activity (MacManus et al., 2000), as well as forming Ca2+-permeable pores in lipid bilayers (Arispe et al., 1993), to increase intracellular Ca2+ concentration as part of the pathogenic mechanism. Aβ also reduces glutamate uptake by astrocytes and increases the activation of glutamate receptors to evoke excitotoxicity (Sonkusare et al., 2005). Thus, strategies that reduce Ca2+ influx and limit excitotoxicity may confer neuroprotection in AD. The non-competitive NMDA receptor antagonist, memantine (Namenda, Ebixa) is used in the treatment of moderate to severe AD (Cosman et al., 2007), and its beneficial properties are based on an ability to inhibit pathological, but not physiological, functions of NMDA receptors, as well as antioxidant action and a propensity to increase production of brain-derived neurotrophic factor in the brain (Sonkusare et al., 2005).
 
Manipulation of the cannabinoid system has several consequences that mirror those observed with memantine. Thus, the protective effects of some cannabinoids are related to the direct regulation of the NMDA receptor, since the non-psychotropic cannabinoid, HU-211, acts as a stereoselective inhibitor of the NMDA receptor and protects rat forebrain cultures (Nadler et al., 1993) and cortical neuronal cultures (Eshhar et al., 1993) from NMDA-induced neurotoxicity.
 
Furthermore, activation of the CB1 receptor protects mouse spinal neurons (Abood et al., 2001) and cultured hippocampal neurones (Shen and Thayer, 1998) from excitotoxicity, possibly through inhibition of presynaptic Ca2+ entry (Mackie and Hille, 1992; Twitchell et al., 1997) and the subsequent suppression of excessive glutamatergic synaptic activity (Shen and Thayer, 1998; Takahashi and Castillo, 2006). CB1 receptor agonists also inhibit glutamate release, which may contribute to a reduction in excitotoxicity (Wang, 2003). The evidence for a Ca2+-dependent synthesis of anandamide and 2-arachidonoyl glycerol (Di Marzo et al., 1994; Stella et al., 1997) would suggest that endocannabinoids are generated in response to an intracellular Ca2+ load in an attempt to provide feedback inhibition of excitotoxicity. In this regard it is notable that endocannabinoid upregulation is a feature of a number of neurotoxic paradigms that are associated with elevated intracellular Ca2+ concentration (Hansen et al., 2001). Alternative mechanisms that are pivotal to cannabinoid-mediated protection include inhibition of [Ca2+]i by reducing calcium release from ryanodine-sensitive stores (Zhuang et al., 2005), inhibition of protein kinase A and reduced nitric oxide generation (Kim et al., 2006).
 
Like memantine, cannabinoids are also capable of increasing brain-derived neurotrophic factor to confer protection against excitotoxicity (Khaspekov et al., 2004). In non-neuronal cells, the induction of nerve growth factor is also facilitated by cannabinoids, acting through the PI3K/PKB pathway (Sanchez et al., 2003), and activation of the CB1 receptor by the endocannabinoid, 2-arachidonoyl glycerol, can also couple to an axonal growth response, whereas CB1 receptor antagonists inhibit axonal growth (Williams et al., 2003). Thus, dampening excessive glutamatergic transmission and excitotoxicity, coupled with neurotrophic actions, may represent interesting actions of cannabinoids that could be exploited for the treatment of AD.

Cannabidiol prevents Aβ-mediated neurotoxicity
 
Cannabidiol (CBD) is the principal non-psychoactive component of Cannabis sativa, with potent antioxidant properties that offer neuroprotection against glutamate toxicity (Hampson et al., 1998). In differentiated PC12 cells exposed to Aβ, CBD reduces the induction of inducible nitric oxide synthase (iNOS), nitric oxide production and activation of the stress-activated protein kinase p38 and the inflammatory transcription factor, nuclear factor-κB (Esposito et al., 2006a), providing evidence for a CBD-mediated downregulation of the inflammatory signalling events associated with exposure to Aβ. As well, CBD reduces Aβ-induced neuronal cell death by virtue of its ability to scavenge reactive oxygen species and reduce lipid peroxidation; antioxidant properties that occur independently of the CB1 receptor (Iuvone et al., 2004).
 
CBD also reverses tau hyperphosphorylation, a key hallmark of AD, by reducing phosphorylation of glycogen synthase kinase-3β, a tau protein kinase responsible for the tau hyperphosphorylation in AD (Esposito et al., 2006b). Moreover, since glycogen synthase kinase-3β also evokes amyloid precursor protein processing to increase Aβ production (Phiel et al., 2003), the CBD-mediated inhibition of glycogen synthase kinase-3β is likely to be effective in reducing the amyloid burden. Thus, from such in vitro studies one can speculate that CBD may be therapeutically beneficial in AD, since it can prevent the deleterious effects of Aβ and ameliorate several features of AD pathology, including tau hyperphosphorylation, oxidative stress, neuroinflammation and apoptosis.
 
Whether such actions of CBD are retained in the AD brain remains to be established, and experiments to test the effect of CBD in the various transgenic animal models of AD are eagerly awaited. In the meantime, reports that CBD is effective as an antioxidant and neuroprotectant in an animal model of Parkinson's disease (Lastres-Becker et al., 2005), and orally effective in a rat model of chronic inflammation (Costa et al., 2007), lend support to its potential therapeutic value in AD.
 
There are a number of advantages of CBD as a therapeutic agent for AD; it is devoid of psychoactive activity and since CB receptors are nitrosylated in the AD brain, a feature that may hinder CB receptors coupling to their downstream effectors (Ramirez et al., 2005), a therapy that does not depend on signalling through CB receptors may have a distinct advantage. Sativex is a cannabinoid-based oromucosal spray, containing CBD and THC, that is devoid of tolerance or withdrawal symptoms (Perez, 2006). This therapy is already available for the treatment of neuropathic pain and multiple sclerosis and may be exploited in the future for the treatment of AD.

CB2 receptors and neuroinflammation
 
The CB2 receptor is largely confined to glial cells in the brain (Nunez et al., 2004), although some studies have reported CB2 receptors in neuronal populations within the brainstem and cerebellum (van Sickle et al., 2005; Ashton et al., 2006). CB2 receptors have been implicated in the control of neural survival (Fernandez-Ruiz et al., 2007) and mediate neuroprotection through their anti-inflammatory actions (Ehrhart et al., 2005). CB2 receptors are upregulated in activated microglia and astrocytes, and this upregulation is proposed to control the local production of proinflammatory mediators such as interleukin-1β, reactive oxygen species and prostaglandins. In the AD brain and in animal models of AD-like pathology, CB2 receptors are upregulated within the active microglia present in those brain regions where senile plaques are abundant (Benito et al., 2003; Ramirez et al., 2005).
 
The upregulation of CB2 in such pathological situations may be an attempt to reduce neuroinflammation since CB2 receptor activation in vitro reduces the microglial production of pro-inflammatory molecules (Facchinetti et al., 2003). Such control in the production of inflammatory mediators may be due to a direct impact on activity of transcription factors, such as nuclear factor κB (Panikashvili et al., 2005; Esposito et al., 2006a).
 
Thus, the neuroprotective mechanisms of cannabinoids are likely to include a downregulation in activity of the transcription factors that are pertinent to induction of the pro-inflammatory cytokines that serve as key players in neurodegenerative disease, while also stimulating the production of anti-inflammatory species such as IL-1ra (Molina-Holgado et al., 2003). The manipulation of such inflammatory pathways may be exploited for the treatment of AD. In support of this contention, Ramirez et al. (2005) have demonstrated that in rats treated with Aβ, the induction of AD-like pathology and cognitive impairment, is reversed by the CB1/CB2 agonist, WIN,55212–22 and the CB2-selective agonist, JWH-133.
 
Since the CB2 receptor was only associated with activated microglia located within the plaque, those authors have suggested that the CB2 receptor may be a promising target for AD by virtue of its ability to serve as a brake for the neuroinflammatory cascade that is a feature of AD. CB2 agonists offer the advantage of being devoid of psychoactivity, although it is important to recognize that they may have other side effects such as immune suppression (Pertwee, 2005), which would be undesirable in an elderly population.

Cannabinoids and neurogenesis in the adult brain
 
Another exciting mechanism that could account for the ability of cannabinoids to confer neuroprotection may be related to their regulation of neurogenesis.
 
Adult neurogenesis can occur in the dentate gyrus of the hippocampus and the subventricular zone (Grote and Hannan, 2007), resulting in the presence of newly generated neurones. In several mouse models of AD neurogenesis is reduced (Dong et al., 2004), although it should be noted that in the post-mortem AD brain, neurogenesis is increased (Jin et al., 2004). Factors that enhance neurogenesis, such as dietary restriction and upregulation of brain-derived neurotrophic factor, enhance neurogenesis and improve the memory performance in animal models of AD (Lee et al., 2000). Thus, targeting adult neurogenesis is receiving interest as a means to mitigate the symptoms of AD. In this regard it is notable that the cannabinoid system also regulates neurogenesis (Galve-Roperh et al., 2007).
 
Adult neurogenesis is defective in mice lacking CB1 receptors (Jin et al., 2004), and the synthetic cannabinoid, WIN55212-2, stimulates adult neurogenesis by opposing the antineurogenic effect of nitric oxide (Kim et al., 2006). Also, the CB1 agonist HU-210 has anxiolytic and antidepressant effects, which may be a functional consequence of enhanced neurogenesis (Jiang et al., 2005). CB2 receptor activation also stimulates neural progenitor proliferation in vitro and in vivo (Palazuelos et al., 2006), and targeting neurogenesis via the CB2 receptor would avoid undesired psychoactive side effects. Thus, the neuroprotective effects of cannabinoids may involve short-term adaptation to neuronal stress, such as limiting excitotoxicity, as well as longer-term adaptations, such as enhancing neurogenesis. It remains to be established whether or not the beneficial effects of cannabinoids on memory, neuroinflammation and neurodegeneration in animal models of AD are due to a functional consequence of an enhancement in neurogenesis.

Targeting acetylcholinesterase with cannabinoids
 
Currently there are four approved drugs (tacrine, Cognex; donepezil, Aricept; rivastigmine, Exelon; galantamine, Reminyl) that are used to alleviate the symptoms of early stage AD by inhibiting the active site of AChE, thus increasing the levels of ACh at the synaptic cleft and enhancing cholinergic transmission.
 
In addition, AChE accelerates that assembly of Aβ peptides into fibrillar species by forming complexes with Aβ via the peripheral anionic site on AChE (Inestrosa et al., 1996), an interaction that increases the neurotoxicity of the Aβ fibrils (Alvarez et al., 1998). Thus, AChE inhibitors offer a two-pronged attack for the treatment of AD by virtue of their ability to enhance ACh availability, as well as reduce amyloidogenesis and subsequent neurotoxicity. A recent study has demonstrated that Δ9-THC competitively inhibits AChE and prevents the AChE-induced aggregation of Aβ by virtue of Δ9-THC binding to the peripheral anionic site on AChE (Eubanks et al., 2006).
 
Compared with tacrine and donepezil, Δ9-THC was found to be more robust inhibitor of Aβ aggregation, suggesting that Δ9-THC and its analogues warrant further investigation as AChE inhibitors for use in the treatment of AD.

Do cannabinoids have a role for the treatment of other neurodegenerative conditions?
 
It is also worth considering how the aforementioned properties of cannabinoids may be beneficial in ameliorating the symptoms of other diseases in which neuroinflammation, oxidative stress and neurodegeneration are key features, such as multiple sclerosis and Parkinson's disease. Benito et al. (2007) have reported that components of the cannabinoid system are upregulated in multiple sclerosis (MS) plaques, suggesting that endocannabinoids either have a role in the pathogenesis of MS or may be upregulated as a consequence of the pathology. MS is associated with excitotoxicity (Pitt et al., 2000; Smith et al., 2000) and neuroinflammation (Ziemssen, 2005), and these represent features of the disease that cannabinoids may be able to circumvent. In encephalomyelitis virus-induced demyelinating disease, an animal model of MS, the mixed cannabinoid agonist HU210 reduces axonal damage and improves motor function as a consequence of a concomitant activation of the CB1 receptor in neurones and CB2 in astrocytes (Docagne et al., 2007).
 
Other studies in animal models of MS have demonstrated a role for the CB2 receptor in enhancing T-cell apoptosis (Sanchez et al., 2006) and suppressing microglial activation (Ehrhart et al., 2005), while the CB1 receptor is associated with neuroprotection (Pryce and Baker, 2007). Such neuroprotective and antioxidant properties of cannabinoids also underlie their ability to reverse the motor deficits in animal models of Parkinson's disease (Lastres-Becker et al., 2005; Garcia-Arencibia et al., 2007), and lend support of a potential role for cannabinoid-based therapies to mitigate the symptoms of a range of neurodegenerative conditions.

Conclusion
 
Alzheimer's disease is a devastating illness for which there is no cure. Current AD drugs, which serve as AChE inhibitors, have a number of unpleasant side effects such as hepatotoxicity and gastrointestinal disturbances. While the NMDA receptor antagonist, memantine, can modify the disease, it cannot reverse the process of neurodegeneration. Manipulation of the cannabinoid pathway offers a novel pharmacological approach for the treatment of AD that may be more efficacious than current treatment regimes.
 
Cannabinoids can reduce the oxidative stress, neuroinflammation and apoptosis that is evoked by Aβ, while promoting the brain's intrinsic repair mechanisms. Certain cannabinoids, such as Δ9-THC, may also increase ACh availability and reduce amyloidogenesis, although potential psychoactive side effects may hinder its clinical usefulness. Cannabinoids clearly offer a multifaceted approach for the treatment of AD and future studies should focus on examining the efficacy of cannabinoids in the array of animal models that exhibit AD-like pathology and cognitive decline.
 
Targeting the CB2 receptor to reduce neuroinflammation while stimulating neurogenesis is likely to be of particular interest, given the reduced risk of psychoactive activity and the close association of the CB2 receptor with the senile plaque, thus limiting drug effects to the region of pathology and sparing the potential for widespread effects on normal neurophysiological processes. In conclusion, manipulation of the cannabinoid system offers the potential to upregulate neuroprotective mechanisms while dampening neuroinflammation. Whether these properties will be beneficial in the treatment of AD in the future is an exciting topic that undoubtedly warrants further investigation (Figure 1).
 
Figure 1
Figure 1
Potential sites of action of the cannabinoid system for the treatment of AD. Activation of the CB2 receptor reduces the formation of reactive oxygen species (ROS) and the release of interleukin-1β from microglia, thus exerting an anti-inflammatory (more ...)
Abbreviations
 
β-amyloid
 
ADAlzheimer's disease
 
CBcannabinoid
 
CBDcannabidiol
 
NMDAN-methyl D-aspartate
 
Δ9-THCΔ9-tetrahydrocannabinol

Notes
Conflict of interest
The authors state no conflict of interest.

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US Patent 6630507 - Cannabinoids as antioxidants and neuroprotectants

Abstract

Cannabinoids have been found to have antioxidant properties, unrelated to NMDA receptor antagonism. This new found property makes cannabinoids useful in the treatment and prophylaxis of wide variety of oxidation associated diseases, such as ischemic, age-related, inflammatory and autoimmune diseases.

 

The cannabinoids are found to have particular application as neuroprotectants, for example in limiting neurological damage following ischemic insults, such as stroke and trauma, or in the treatment of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and HIV dementia. Nonpsychoactive cannabinoids, such as cannabidoil, are particularly advantageous to use because they avoid toxicity that is encountered with psychoactive cannabinoids at high doses useful in the method of the present invention. A particular disclosed class of cannabinoids useful as neuroprotective antioxidants is formula (I) wherein the R group is independently selected from the group consisting of H, CH3, and COCH3. ##STR1##

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Cannabidiol in vivo blunts β-amyloid induced neuroinflammation by suppressing IL-1β and iNOS expression

Pharmacological inhibition of beta-amyloid (Aβ) induced reactive gliosis may represent a novel rationale to develop drugs able to blunt neuronal damage and slow the course of Alzheimer's disease (AD). Cannabidiol (CBD), the main non-psychotropic natural cannabinoid, exerts in vitro a combination of neuroprotective effects in different models of Aβ neurotoxicity. The present study, performed in a mouse model of AD-related neuroinflammation, was aimed at confirming in vivo the previously reported antiinflammatory properties of CBD.

Experimental approach:
 
Mice were inoculated with human Aβ (1–42) peptide into the right dorsal hippocampus, and treated daily with vehicle or CBD (2.5 or 10 mg kg−1, i.p.) for 7 days. mRNA for glial fibrillary acidic protein (GFAP) was assessed by in situ hybridization. Protein expression of GFAP, inducible nitric oxide synthase (iNOS) and IL-1β was determined by immunofluorescence analysis. In addition, ELISA assay of IL-1β level and the measurement of NO were performed in dissected and homogenized ipsilateral hippocampi, derived from vehicle and Aβ inoculated mice, in the absence or presence of CBD.
 
Key results:
 
In contrast to vehicle, CBD dose-dependently and significantly inhibited GFAP mRNA and protein expression in Aβ injected animals. Moreover, under the same experimental conditions, CBD impaired iNOS and IL-1β protein expression, and the related NO and IL-1β release.
Conclusion and implications:
 
The results of the present study confirm in vivo anti-inflammatory actions of CBD, emphasizing the importance of this compound as a novel promising pharmacological tool capable of attenuating Aβ evoked neuroinflammatory responses.
Keywords: cannabidiol, Aβ, reactive gliosis, GFAP, IL-1β, iNOS, mice

Introduction
 
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder (Koo et al., 1999) whose specific hallmarks are neurofibrillary tangles (Terry, 1963) and senile plaques (Braak and Braak, 1997). While neurofibrillary tangles result from the deposition of hyperphosphorylated tau proteins (Lee et al., 1991), senile plaques represent more complex extracellular lesions composed of a core of β-amyloid (Aβ) aggregates, surrounded by activated astrocytes and dystrophic neuritis (Itagaki et al., 1989; Cotman et al., 1996).
 
At present, although biochemical events leading to Aβ neurotoxicity still remain unclear, proposed mechanisms include production of oxygen free radicals (Behl et al., 1994), changes in cytosolic calcium homeostasis (Ueda et al., 1997; Mattson, 2002) and activation of Wnt pathway as well as of the transcription nuclear factor NF-κB (Green and Peers, 2002; Caricasole et al., 2003). In addition to cytotoxic mechanisms directly affecting neurons, Aβ-induced glial cell activation, triggering inflammatory responses with subsequent release of neurotoxic cytokines, is present in the AD brain, contributing to the pathogenesis of disease (Craft et al., 2006). The possibility of interfering with this detrimental cycle by pharmacologically inhibiting reactive gliosis has been proposed as a novel rationale to develop drugs able to blunt neuronal damage and consequently slow the course of disease.
 
Cannabidiol (CBD), the main non-psychotropic component of the glandular hairs of Cannabis sativa, exhibits a plethora of actions including anti-convulsive, sedative, hypnotic, anti-psychotic, anti-nausea, anti-inflammatory and anti-hyperalgesic properties (Mechoulam et al., 2002; Costa et al., 2007). CBD has been proved to exert in vitro a combination of neuroprotective effects in Aβ-induced neurotoxicity, including anti-oxidant and anti-apoptotic effects (Iuvone et al., 2004), tau protein hyperphosphorylation inhibition through the Wnt pathway (Esposito et al., 2006a), and marked decrease of inducible nitric oxide synthase (iNOS) protein expression and nitrite production in Aβ-challenged differentiated rat neuronal cells (Esposito et al., 2006b).
In spite of the large amount of data describing the significant neuroprotective and anti-inflammatory properties of CBD in vitro, to date no evidence has been provided showing similar effects in vivo. To achieve this, the present study investigated the potential anti-inflammatory effect of CBD in a mouse model of AD-related neuroinflammation induced by the intrahippocampal injection of the human Aβ (1–42) fragment.

Methods
 
Animal care
Experiments were conducted in 3–5-months old C57BL/6J mice (35–40 g) (Harlan, Udine, Italy). Animals were housed under controlled illumination (12 h light/12 h dark cycle; light on 0600h) and standard environmental conditions (ambient temperature 20–22°C, humidity 55–60%) for at least 1 week before starting experiments. Food and water were available ad libitum. All surgery and experimental procedures were performed during the light cycle and were made according to the National Institutes of Health guidelines for the care and use of laboratory animals and to those of the Italian Ministry of Health (DL 116/92), and were approved by the local Institutional Animal Care and Use Committees. All efforts were made to reduce both animal number and suffering during the experiments.
 
Surgical preparation
 
Mice were anaesthetized with halothane (1–3%), placed in a stereotaxic frame, and injected with 10 ng of Aβ (1–42) (Tocris Cookson, Bristol, UK) or vehicle artificial cerebrospinal fluid (aCSF) into the right dorsal hippocampus, using the following coordinates relative to the bregma: AP=+2.0 mm; ML=−1.8 mm; DV=−2.3 mm. The flow was maintained at a constant value of 0.5 μl min−1, using a microdialysis pump and the needle was left in place for additional 5 min to allow for diffusion. Animals were kept on a warming pad until they had fully recovered from the anaesthetic and were kept in individual cages to prevent damage to the scalp sutures until they were killed for tissue processing.
 
Starting on the third day after surgery, mice were intraperitoneally (i.p.) treated daily with vehicle (Tocrisolve 100, Tocris Cookson) or CBD (Tocris Cookson) (2.5 or 10 mg kg−1) for 7 days.
 
The doses of the drug were selected according to previous literature (Mechoulam et al., 2002), whereas the i.p. administration route was derived from the author's experience in such animal model of AD. Animals for in situ hybridization analysis were killed by cervical dislocation and brain were removed, snap-frozen on dry-ice, and stored at −80°C. Brains were mounted on Tissue Tek (Polysciences, PA, USA), and 14-μm-thick coronal sections were cut on a cryostat Microtome HM560 (Microm, Walldorf, Germany). Sections were mounted onto frozen SuperFrost/Plus slides (Fisher Scientific, Schwerte, Germany), dried on a 42°C warming plate, and stored at −20°C until used. Animals for immunofluorescence analysis were killed and perfused with HEPES buffer containing protease inhibitors; brains were rapidly frozen in liquid N2.
 
Tissue was cut on a freezing sliding microtome (Leica SM 2000 R, Milan, Italy) to obtain 30 μm sections collected in a 15 mM NaN3 phosphate-buffered saline (PBS) solution and stored at 4°C. For enzyme-linked immunosorbent assay (ELISA) experiments brains were bisected down the sagittal sulcus and the hippocampus was dissected out of the right side and quickly frozen in liquid N2.
In situ hybridization
 
Sections were fixed in ice-cold 4% paraformaldehyde for 20 min, rinsed in PBS, quenched for 15 min in 1% H2O2 methanol solution, rinsed in PBS, quenched for 8 min in 0.2 M HCl, rinsed in PBS, treated with proteinase K 20 μg ml−1 (Roche Molecular Diagnostics, Milan, Italy) in 50 mM Tris-HCl, 5 mM ethylene diamine tetra acetic acid (EDTA) (pH 8.0) for 10 min, rinsed in PBS, fixed in ice-cold 4% paraformaldehyde, incubated for 10 min in 0.1 M triethanolamine (pH 8.0) to which 1.2 ml acetic anhydride was added dropwise, rinsed in PBS, washed with 0.9% NaCl for 5 min, dehydrated in graded series of ethanol and air-dried. Hybridization was carried out in 100 μl of hybridization buffer containing specific sense or antisense 35S-labelled riboprobe for glial fibrillary acidic protein (GFAP; 70 000–100 000 c.p.m. μl−1). Hybridization buffer consisted of 50% deionized formamide, 20 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 5 mM EDTA (pH 8.0), 10% dextran sulphate (Sigma, Milan, Italy), 0.02% Ficoll 400 (Sigma), 0.02% polyvinylpyrrolidone (PVP 40; Sigma), 0.02% bovine serum albumin (BSA; Sigma), 0.5 mg ml−1 tRNA (Roche Molecular Diagnostics), 0.2 mg ml−1 fragmented herring sperm DNA and 200 mM dithiothreitol.
 
Before applying to the tissue the hybridization cocktail was denatured for 2 min at 95°C. Slides were incubated overnight at 54°C in a humidified chamber. Four high-stringency washes were carried out at 62°C with 5 × saline sodium citrate (SSC)/0.05% Tween-20 (Sigma), then with 50% formamide/2 × SSC/0.05% Tween-20, with 50% formamide/1 × SSC/0.05% Tween-20 and finally with 0.1 × SSC/0.05% Tween-20.
 
Slides were dehydrated in graded ethanol series, air-dried and exposed to Biomax MR film (Scientific Imaging Systems, NY, USA). GFAP mRNA expression was semi-quantified by densitometric scanning of the Biomax film with a GS 700 imaging densitometer (Bio-Rad Laboratories, CA, USA) and a computer programme (Molecular Analyst, IBM, Milan, Italy).
 
Immunofluorescence
 
Brain coronal sections (30 μm) were fixed for 30 min in 4% paraformaldehyde, washed with PBS, and blocked for 15 min with 10% BSA. Sections were then incubated for 2 h with one of the following primary antibodies: monoclonal anti-GFAP (1:200, Lab Vision, CA, USA), monoclonal anti-IL-1β (1:100, Sigma) and monoclonal anti-iNOS (1:100, Sigma).
 
Following PBS washing, sections were incubated in the dark for half an hour with Texas Red-conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:200; AbCam, Cambridge, UK). After final PBS washing, sections were analysed with a Zeiss LSM 410 microscope equipped with a krypton/argon laser, dichroic beam splitters and barrier emission filters needed for triple labelling. Texas Red was excited at a wavelength of 568 nm and collected through a long pass filter (590LP). FITC was excited with a wavelength of 488 nm and collected with a narrow band filter (515–540BP). Texas Red and FITC were assigned to the red and green channels respectively of the generated RGB image.
Nitrite assay
 
NO was measured as nitrite (NO2) accumulated in the inoculated ipsilateral hippocampi. A spectrophotometer assay based on the Griess reaction was used (Di Rosa et al., 1990). Briefly, Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine in H3PO4) was added to an equal volume of homogenized tissue supernatant and the absorbance at 550 nm was measured after 10 min. The NO2 concentration was thus determined using a standard curve of NaNO2 and referred to μg of homogenized hippocampal protein content according to BioRad assay method.
IL-1β assay
 
ELISA was used to quantify the presence of IL-1β in the supernatant of homogenized hippocampi ipsilateral to the inoculation site. A mouse IL-1β ELISA kit (R&D System, MN, USA) was used according to the manufacturer's recommendations. Briefly, 50 μl of standard, control buffer, or sample was combined with 50 μl of assay buffer in IL-1β antibody-coated wells on the ELISA plate and incubated at room temperature for 2 h. Wells were washed five times before the addition of 100 μl of the appropriate horseradish peroxidase conjugate and incubated for 2 h more.
 
After a second wash cycle, 100 μl of hydrogen peroxide/tetramethylbenzidine substrate solution was added per well, and the plate was incubated for 30 min at room temperature in the dark. The reaction was stopped by addition of the hydrochloric acid solution provided in the kit. The absorbance at 450 nm was measured with a microreader (Bio-Rad Laboratories, 3550-UV) with wavelength correction at 570 nm.
 
Statistical analysis
 
Results were expressed as mean±s.e.m. of experiments. Statistical analysis was performed using analysis of variance, and multiple comparisons were performed by Bonferroni's test, with P<0.05 considered significant.

Results
 
CBD effects on GFAP mRNA expression in Aβ inoculated mice
 
The induction of mRNA for GFAP protein 10 days following intrahippocampal injection of Aβ (1–42) (10 μg ml−1) was examined. As shown in Figure 1, GFAP mRNA, as measured by densitometry, was significantly increased by Aβ treatment in comparison with mice hippocampi injected with vehicle (+883±12%). CBD (2.5 or 10 mg kg−1) dose-dependently and significantly inhibited (−31.3±4.1 and −81±6.7% respectively) GFAP mRNA expression versus Aβ-injected animals i.p. treated with vehicle. Negligible or no increase in GFAP mRNA was observed following treatment with Aβ (42–1) reverse peptide or CBD alone (data not shown).
 
Figure 1
Figure 1
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on glial fibrillary acidic protein (GFAP) mRNA in mouse hippocampus. Upper panel: Dark-field photomicrographs showing the distribution of GFAP mRNA as detected by (more ...)
 
 
CBD effects on GFAP, iNOS and IL-1β protein expression in Aβ inoculated mice
Immunofluorescence analysis was aimed at estimating the effect of CBD treatment on the expression of inflammatory proteins 10 days following Aβ (1–42) (10 μg ml−1) injection into mouse hippocampi.
 
As shown in Figures 2, ,33 and and4,4, the number of GFAP, iNOS and IL-1β-positive cells was significantly increased by Aβ (+407±34, +1025±68, and +1288±16% respectively) versus vehicle-inoculated hippocampi. CBD (2.5 or 10 mg kg−1) treatment dose-dependently and significantly inhibited the number of cells positive for GFAP (−30±3.12 and −64.14±6.2% respectively), iNOS (−33.3±5.2 and −61.5±4.25% respectively) or IL-1β (−30.5±5.7 and −68±4.23% respectively), in comparison with animals given Aβ and injected with CBD vehicle. Also in this case, negligible or no increase in GFAP, iNOS and IL-1β was observed following treatment with Aβ (42–1) reverse peptide or CBD alone (data not shown).
 
Figure 2
Figure 2
 
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on glial fibrillary acidic protein (GFAP) in mouse hippocampus. Upper panel:Representative photomicrographs showing GFAP immunoreactive cells in: (a) vehicle inoculated (more ...)
Figure 3
Figure 3
 
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on inducible nitric oxide synthase (iNOS) in mouse hippocampus. Upper panel: Representative photomicrographs showing iNOS immunoreactive cells in: (a) vehicle inoculated (more ...)
Figure 4
 
 
Effects of cannabidiol (CBD) (intraperitoneal (i.p.) treatment for 7 consecutive days) on IL-1β in mouse hippocampus. Upper panel: Representative photomicrographs showing IL-1β immunoreactive cells in: (a) vehicle inoculated mice (control), (more ...)
 
CBD effects on NO release in hippocampal homogenates
 
The release of NO was evaluated by measurement of its stable metabolite (NO2) in homogenized ipsilateral hippocampi 10 days after Aβ (1–42) (10 μg ml−1) injection. As shown in Figure 5 NO2 levels were significantly increased by Aβ injection in comparison with vehicle-inoculated hippocampi (+525±30%). CBD (2.5 or 10 mg kg−1) treatment dose dependently and significantly inhibited NO2 release in tissue homogenates (−30±1 and −51±3.71% respectively) compared with those from mice injected with vehicle.
 
Figure 5
 
Effects of cannabidiol (CBD) (2.5 or 10 mg kg−1 intraperitoneal (i.p.) for 7 consecutive days) on nitrite (NO2) level in mouse hippocampal homogenates 10 days after Aβ (1–42) (10 μg ml (more ...)
CBD effects on IL-1β levels in hippocampal homogenates
ELISA assay was performed on homogenized ipsilateral hippocampi 10 days following Aβ (1–42) (10 μg ml−1) injection to evaluate the effect of CBD treatment on IL-1β release. As shown in Figure 6, IL-1β level was significantly increased by Aβ injection in comparison with vehicle-inoculated hippocampi (+900±60%). Treatment with CBD (2.5 or 10 mg kg−1) dose-dependently and significantly inhibited IL-1β release in tissue homogenates (−30±3 and −46.7±4% respectively) when compared with homogenates derived from vehicle-treated animals.
Figure 6
 
 
Effects of cannabidiol (CBD) (2.5 or 10 mg kg−1 intraperitoneal (i.p.) for 7 consecutive days) on IL-1β level in hippocampal homogenates 10 days after Aβ (1–42) (10 μg ml−1 (more ...)
 
Discussion and conclusions
 
The urgent need for novel strategies for AD is apparent with the realization that the currently approved therapies are only palliative without significant and substantial disease modifying effects (Turner, 2006). In contrast, the present study suggests that CBD, here investigated with a primary focus on glial pathways, exhibits a potential to delay effectively the onset and progression of Aβ neurotoxicity. Actually, the current results provide evidence that CBD causes a clear-cut reduction of the transcription and expression of glial pro-inflammatory molecules in the hippocampus of an in vivo model of Aβ-induced neuroinflammation.
 
They suggest CBD may be regarded as a promising tool able to affect the course of Aβ-related neuropathology, by reducing Aβ-generated reactive gliosis and subsequent neuroinflammatory responses, in addition to the previously demonstrated protective effects directly affecting neurons (Iuvone et al., 2004; Esposito et al., 2006a, 2006b).
 
Indeed, the increasing body of immunohistological and molecular findings, showing that inflammatory processes are pre-eminent and constant aspects of the neuropathology generated by the Aβ toxicity, supports the notion that the previously under-appreciated glial activation plays a critical role in the pathogenesis of brain lesions subsequent to Aβ deposition (Craft et al., 2006). Although acute activation of glial cells may have important beneficial effects in the recovery of the CNS from a variety of insults, it is believed that a persistent activation amplifies inflammatory responses leading to a worsening of the consequences of injury (Ralay Ranaivo et al., 2006). In the scenario of reactive gliosis, the main features are astrocytic hypertrophy and proliferation, along with a marked overexpression of the intermediate filament proteins, such as GFAP, the best known hallmark of activated astrocytes (O'Callaghan and Sriram, 2005).
 
The present investigation focuses the ability of this phytocannabinoid, CBD, to negatively modulate GFAP transcription and expression as well as to significantly reduce IL-1β and iNOS upregulation, which importantly contribute to disease progression, through the propagation of inflammation and oxidative stress. Among the many active substances produced by Aβ stimulated microglia, IL-1β has proved to be substantially implicated in the cytokine cycle of cellular and molecular events responsible for the neurodegenerative consequences (Griffin et al., 1998). These include synthesis and processing of amyloid precursor protein (Buxbaum et al., 1992; Mrak and Griffin, 2000), as well as astrocyte activation with a subsequent iNOS overexpression and excessive production of NO (Das and Potter, 1995; Sheng et al., 1996). Increasing amounts of NO, a short-lived and diffusible free radical involved in all reported neuroinflammatory and neurodegenerative conditions (Murphy, 2000), accelerate neuronal protein nitration and cause a marked increase in tau protein hyperphosphorylation (Saez et al., 2004), encouraging the detrimental progression of Aβ-related pathology (Nathan et al., 2005).
 
Therefore, in this context where inflammatory pathways are believed to play relevant roles as driving forces of the Aβ-induced injury, they are identified as potential modulators of the neuronal damage and are reported as neuronal targets for effective therapeutic interventions. The present investigation provides the first evidence that substantial components of the neuroinflammatory response, set in motion by Aβ deposition and allowing for progression of neuropathology, are suppressed in vivo by CBD. The current data confirm and further reinforces the view that CBD can exhibit protective effects in models of neuroinflammation/neurodegeneration.
 
The seminal work describing CBD neuroprotective properties demonstrated its ability to protect cortical neurons in culture against glutamate-induced neurotoxicity. Such effects were found to be not antagonized by the established CB1 antagonist SR141716A, suggesting that they were independent of CB1 cannabinoid receptor involvement. Later CBD was shown to prevent Aβ-induced toxicity in PC12 pheocromocytoma cells, increasing survival while decreasing reactive oxygen species production, lipid peroxidation, caspas-3 levels, DNA fragmentation and intracellular calcium (Iuvone et al., 2004).
 
In addition to this combination of anti-oxidant, anti-inflammatory and anti-apoptotic effects, subsequent studies, carried out under the same experimental conditions, demonstrated that CBD was able to operate as a Wnt/β-catenin pathway rescuer, inhibiting Aβ-induced tau protein hyperphosphorylation while attenuating iNOS protein expression and NO production (Esposito et al., 2006b). Such a wide range of effects on pathophysiological processes implicated in neuroinflammatory/neurodegenerative diseases appears truly intriguing and encourages the clinical applicability of CBD for therapeutic use.
 
Its antioxidant and neuroprotective actions are presumably related in part to a potential as a scavenger of free radicals due to its structural characteristics (Hampson et al., 2000), although there is room for alternative mechanisms. Ruling out the possibility that transient receptor potential vanilloid type 1 channels may be involved in the suppression of reactive gliosis exerted by CBD (personal data), a potential involvement of the CB2 receptor might be taken into account. The recently provided in vitro evidence that CBD can display CB2 receptor inverse agonist properties (Thomas et al., 2007) might offer an explanation of the anti-neuroinflammatory effects we have shown here. In Aβ neurotoxicity, several results have related CB2 receptors to events involved in the progression of brain damage by affecting reactive gliosis at neuroinflammatory lesion sites (Walter and Stella, 2004). Further, we have recently reported that, in a rodent model of Aβ-induced reactive gliosis, CB2 receptors were overexpressed (van der Stelt et al., 2006), paralleling the changes in cannabinoid receptor expression occurring in AD brain, where, in astrocyte-associated plaques, CB2 receptors were also found to be up-regulated (Ramirez et al., 2005). Interestingly, some of our recent unpublished results suggest that pharmacological interactions at glial CB1 and CB2 receptors result in a marked and opposite regulation of reactive astroglial response, with CB2 receptor blockade suppressing astroglial activation.
 
These findings would imply a function for CB2 receptors in the regulation of CBD actions and would encourage further study of how pharmacological interactions at this receptor could influence the effects of CBD. Although more research will be needed to elucidate fully the molecular mechanisms implicated in the CBD actions described in this paper, the current data showed that the early administration of CBD markedly attenuated in vivo the reactive gliosis induced by Aβ injury. The relevance of these results stems from the fact that a proper control of glial cell function, which is compromised by the persistence of inflammatory events, is critical to provide an environment capable of ensuring neuronal survival and function. For this reason, on the basis of the present results, CBD, a drug well tolerated in humans, may be regarded as an attractive medical alternative for the treatment of AD, because of its lack of psychoactive and cognitive effects.
 
Acknowledgments
 
This work was supported by FIRB2006.
Abbreviations
 
Aβbeta amyloid
 
ADAlzheimer's disease
 
CBDcannabidiol
 
i.p.intraperitoneally
 
GFAPglial fibrillary acidic protein
 
iNOSinducible nitric oxide synthase
 
IL-1βinterleukin 1 beta
 
ELISAenzyme linked immunosorbent assay

Notes
Conflict of interest
The authors state no conflict of interest.

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Inflammation and aging: can endocannabinoids help?

Biomed Pharmacother. Author manuscript; available in PMC 2009 April 1.
Published in final edited form as:
PMCID: PMC2408719
NIHMSID: NIHMS51644
 
Inflammation and aging: can endocannabinoids help?
Yannick Marchalant, Holly M Brothers, and Gary L Wenk
 Department of Psychology, Psychology building, The Ohio State University, Columbus, OH, 43210 USA
 
 
Yannick Marchalant: [email protected], Holly M Brothers: [email protected], Gary L Wenk: [email protected]
 
 
Summary
 
Aging often leads to cognitive decline due to neurodegenerative process in the brain. As people live longer, a growing concern exist linked to long-term, slowly debilitating diseases that have not yet found a cure, such as Alzheimer’s disease. Recently, the role of neuroinflammation has attracted attention due to its slow onset, chronic nature and its possible role in the development of many different neurodegenerative diseases.
In the future, treatment of chronic neuroinflammation may help counteract aspects of neurodegenerative disease. Our recent studies have focused upon the endocannabinoid system for its unique effects on the expression of neuroinflammation. The basis for the manipulation of the endocannabinoid system in the brain in combination with existing treatments for Alzheimer’s disease will be discussed in this review.

Alzheimer’s disease (AD) is the most common neurodegenerative disease and accounts for the majority of diagnosed dementia after age 60. It is estimated to currently affect between 20 and 30 million people worldwide, with 4 million in the U.S. alone (Selkoe, 2005).
 
The prevalence of the disease increases greatly after 60 years of age (1–3%) to reach around 30% of the population at 85 years old and up (Walsh and Selkoe, 2004). As life expectancy increases in developed countries, the incidence of AD, and its burden on healthcare, is very likely to increase dramatically in the next few decades.
 
Two classes of drugs are currently used in the AD symptomatology: acetylcholinesterase inhibitors (Donopezil, Rivastigmine, Galantamine) and a single N-methyl-D-asparate (NMDA) receptor antagonist (Memantine). The first class of drugs tries to maintain brain levels of acetylcholine, known to decrease early on in AD, during the mild to moderate stages of the disease. The second category of drugs regulates post-synaptic calcium ion influx due to the action of glutamate at NMDA receptors and is recommended for use during the moderate to severe AD stages (Lleo et al., 2006, Parsons et al., 2007). Currently available drugs do not reverse or stop the progression of the disease but only relieve certain cognitive symptoms.
 
Therapies currently under investigation either target amyloid protein (Schenk et al., 2005) or pro-inflammatory cytokines such as tumor necrosis factor-alpha (Tobinick and Gross, 2008). The complex nature of AD would advocate for the use of a multimodal drug approach that would also provide protection from the processes that underlie neurodegeneration. In the following paragraphs we will discuss the growing evidence supporting a role for the endocannabinoid system in the regulation of chronic brain inflammation associated with AD and the potential benefits to modulate that system in combination with memantine.

Endocannabinoids
 
Cannabinoid refers to naturally occurring or synthetic molecules mimicking the activity of plant-derived cannabinoids from Cannabis Sativa. Two types of cannabinoid receptors have been so far identified in the body (see Howlett, 2002 for review), named CB1 and CB2 (Matsuda et al., 1990, Munro et al., 1993).
 
Discovery of cannabinoid receptors (CBr) lead to the finding of endogenous agonists for these receptors called endocannabinoids (EC). EC are derived from arachidonic acid, arachidonoylethanolamide (anandamide), and 2-arachidonoyl glycerol (2-AG), synthesized on-demand post-synaptically and released in response to the entry of calcium ions (see Di Marzo et al., 2005 for review).
 
These EC in combination with the two known CBr constitute the endocannabinoid system (ECS). In the central nervous system (CNS), CB1 is overwhelmingly represented over CB2 and particularly abundant in cortical regions, the hippocampus, cerebellum and basal ganglia (Herkenham et al., 1991) while CB2 may be restricted to microglia (Nunez et al., 2004) or neurons in the brainstem (Van sickle et al., 2005) and cerebellum (Ashton et al., 2006). Deactivation of the EC is due to a rapid enzymatic degradation in the synaptic cleft or after membrane transport (Piomelli, 2003). The ECS is thought to be a neuromodulator (Vaughan and Christie, 2005) and an immunomodulator (Klein, 2005).
 
In the CNS, the ECS can influence food intake, endocrine release, motor control, cognitive processes, emotions and perception. Cannabinoids treatment has been shown to be neuroprotective under many experimental conditions.
 
Drugs that manipulate the ECS are currently evaluated in various diseases ranging from cancer to AIDS (Prentiss et al., 2004; Walsh et al., 2003) for their peripheral analgesic and immunosuppressive properties. Their anti-inflammatory actions may make them useful in the treatment of multiple sclerosis, Parkinson’s disease and AD (Maresz et al., 2005; Ramirez et al., 2005; Eljaschewitsch et al., 2006; Marchalant et al., 2007, 2008).
 
Very little in vivo evidence to support the use of EC receptor agonists has been reported (Ramirez et al., 2005; Marchalant et al., 2007, 2008), although in vitro studies have found evidence for their anti-inflammatory effectiveness (Facchinetti et al., 2003; Ramirez et al., 2005; Sheng et al., 2005). Our recent work demonstrated the anti-inflammatory effect of a chronic treatment of a low dose of the CBr agonist WIN-55,212-2 (without psychoactive effects) on the consequences of chronic neuroinflammation induced by the infusion of LPS into the 4th ventricle of young rats (Marchalant et al., 2007).
 
Moreover, that same anti-inflammatory effect was found using a non-psychoactive dose given by slow subcutaneous infusion of WIN-55,212-2 to healthy aged rats; these rats also demonstrated improved spatial memory (Marchalant et al., 2008). Our ongoing work in aged rats has shown that treatment with the CBr agonist WIN-55,212-2 increases neurogenesis in the hippocampus (Unpublished results). Our preliminary data suggest that the neurogenic and anti-inflammatory effects in aged rats are due to the agonist/antagonist properties of WIN-55,212-2 at multiple receptors (Unpublished results).

Neuroinflammation and Alzheimer’s disease
 
Microglial cells and macrophages are key elements of the brain inflammatory response during neurodegenerative diseases such as AD. Microglia’s morphology in its “resting” state is highly ramified and possesses a down-regulated phenotype (low or no detectable expression of cell-surface proteins such as MHC class I or II).
 
Even during that so called “resting” state, those cells are highly involved in surveying their environment using their numerous cellular processes (Nimmerjahn et al., 2005). This resting state seems to be maintained by intercommunication between CD200 produced by neurons and CD200 receptor expressed on microglia (Hoek et al., 2000). We speculate that the loss of this equilibrium could lead to a chronic activation of glia such as seen during aging or associated with neurodegenerative diseases.
 
Subtle changes in the brain homeostasis can then lead to a rapid morphological change and protein expression pattern of those microglia cells (to a pro- or anti-inflammatory response profile) depending on the nature of the brain injury and the stage of the inflammatory response (Stout et al., 2005). In the brains of patients with AD an atypical inflammatory response can be observed characterized by an activation of resident microglia possibly accompanied by monocyte infiltration from the blood (Akiyama et al, 2000). Those activated cells are often found surrounding amyloid plaques either due to the presence of amyloid itself or the presence of neurodegeneration.
 
Chronic neuroinflammation contributes to the pathophysiology of AD (Akiyama et al., 2000; Wenk et al., 2000). Long term use of anti-inflammatory drugs has been hypothesized to counteract with these processes and thus protect against the disease; epidemiological evidence supports the long term use of NSAIDs to reduce the prevalence of AD (Andersen et al., 1995; In’T Veld et al., 1998). Indeed, NSAID use for more than two years was found to be significantly associated with a reduced risk of AD (Breitner et al., 1994; In’T Veld et al., 1998). However, clinical trials so far have produced mostly negative results. Recent studies suggest that anti-inflammatory agents may have a preventative influence on the development of AD pathology even if they do not appear to slow progression of dementia (Breitner et al., 1994; Wenk et al., 2000).
 
Inflammatory processes are closely associated to the neuropathological and cognitive syndromes of AD (Akiyama et al., 2000). Post-mortem analysis of inflammatory markers (activated microglia cells) of AD patient’s samples of the entorhinal and frontal cortex clearly correlated with synaptic loss in those regions.
 
That correlation between inflammation and cell loss was even greater than between cell loss and neurofibrillary tangle density (DiPatre and Gelman, 1997) or degree of deposition of amyloid (Terry et al., 1991). It is not surprising therefore that the cascade of immunological events that can be observed in the brain of an AD patient are found to occur very early in the progression of the disease in those same brain regions that later show the greatest concentration of senile plaques and atrophy (Cagnin et al., 2001).
 
Moreover, the development of inflammation within neuronal populations and regions known to be vulnerable in the brains of AD coincide with the memory impairments observed in the early stages of AD pathology (Davis et al., 1999). The brain’s inflammatory response leads to a cascade of self-perpetuating cellular events including increased release of prostaglandins (Katsuura et al., 1989), enhanced release of glutamate (Emerit et al., 2004), and blockade of glutamate uptake by glia (Rothwell et al., 1997). Inflammation is also known to be able to relieve the NMDA channels from their magnesium ion blockade and increase nitric oxide levels, both inducing a dysregulation of calcium ion influx at the post-synaptic membrane.
 
Any subsequent activation of NMDA receptors by glutamate may then enable a continuous entry of calcium ions into neurons, potentially overcoming the endogenous mechanisms regulating calcium ion homeostasis (Albin and Greenamyre, 1992; Chao and Hu, 1994). This unbalance in calcium influx can thus impair mitochondrial respiration, oxidative stress, as well as energy production and membrane depolarization (Emerit et al., 2004). The consequences of long-term, low level brain inflammation might therefore contribute to impairment of calcium homeostasis and alter its downstream signal-transduction cascades (Barry et al., 2005).

Endocannabinoids and Alzheimer’s disease: a useful tool in addition to existing medication?
As described in the previous paragraphs, there is strong evidence that inflammation contributes to the evolution of the AD pathology and that the ECS might be of some use in treating chronic inflammation observed in AD.
 
More classically, AD is described mainly by two post-mortem histological diagnostic features that are extracellular amyloid deposition and Tau hyperphosphorylation forming intracellular neurofibrillary tangles (LaFerla et al., 2007; Walsh and Selkoe, 2007; Kuret et al., 2005). The amyloid plaques forming during AD are due to accumulation of non-soluble fragments of the amyloid protein in various region of the brain, notably the hippocampus, cortex and amygdala. These plaques are well described as part of the neurodegenerative process involved in AD (LaFerla and Oddo, 2005). Amyloid protein can also trigger inflammatory processes as well as long term changes in the NMDA receptor activity, and thus the control of calcium ion influx on the post-synaptic side, to negatively impact the function of those cells (Fiala et al., 2007; Wenk, 2006). Tau protein, a microtubule associated protein, when hyperphosphorylated can accumulate and form neurofibrillary tangles that in turn impair intra-neuronal communication (Ballatore et al., 2007).
 
There is a growing amount of evidence of the possible implication of the ECS in the regulation of events occurring during the course of AD progression, particularly on the regulation of amyloid clearance and inflammation in vitro as well as in vivo (Campbell and Gowran, 2007; Benito et al., 2007). Post-mortem analysis of AD brains demonstrated an increased expression of CBr located on microglia within the amyloid rich plaques (Ramirez et al., 2005) although that seems to not be the case in other studies (Benito et al., 2003). An overall decrease of cortical CB1 receptor seems to occur during AD further away from the plaques (Ramirez et al., 2005); a finding that is similar to that seen in aged rats (Marchalant et al., 2008) with region-specific variability (Liu et al., 2003).
 
Moreover, up-regulation of the fatty acid amide hydrolase occurs within plaques and might be responsible for increase in metabolites from anandamide degradation, such as arachidonic acid, and thus contribute to the inflammatory process seen in AD. CB1 receptor stimulation demonstrated interesting neuroprotective properties (Marsicano et al., 2003) and thus suggests that the ECS might influence neuronal survival and may offer protection from pathological process like amyloid protein in AD. Amyloid infusion in vivo is associated with gliosis and memory impairment; both effects were reversed by infusion of an inhibitor of EC reuptake (Van der Stelt, 2006). The use of an agonist of CBr, WIN-55,212-2, also proved to be effective in reversing memory impairment following i.c.v infusion of amyloid beta, possibly linked to prevention of activation of microglia (Ramirez et al., 2005).
 
We’ve shown that WIN-55,212-2 can reduce the number of activated microglia produced by the long-term infusion of LPS into the 4th ventricle of young rats (Marchalant et al., 2007) as well as the naturally occurring microglia activation associated with normal aging in rats (Marchalant et al., 2008). Taken together the results of these studies using different experimental animal models strongly suggest a potential benefit of the ECS manipulation on the consequences and expression of brain inflammation associated with normal aging and AD. These results thus advocate for an investigation of the effects of ECS stimulation on the presence of brain inflammation in currently available transgenic mouse models of AD.
 
Our work also suggests that if EC have an influence on chronic neuroinflammation in young or aged animals, those effects might not only be due to direct activity of the ECS on microglia cells but also on the demonstrated modulation of glutamatergic transmission by EC in the hippocampus (Haller et al., 2007; Nemeth et al., 2008). The indirect influence of EC receptor stimulation on inflammation through modulation of glutamate release has striking parallels with the action of the memantine, a drug currently approved for the treatment of symptoms in moderate to severe AD patients (Parsons et al., 2007).
 
Memantine is a low to moderate affinity antagonist of the NMDA receptor channel that is able to restore the glutamatergic homeostasis by reducing the signal to noise ratio in presence of excessive synaptic glutamate release in pathophysiological conditions (Parsons et al., 2007). Efficiency of memantine in the symptomatological treatment of AD has been confirmed by clinical studies and additional benefits seems to result from its combined use with cholinesterase inhibitors (Ditzler, 1991; Gortelmeyer and Erbler, 1992; Orgogozo et al., 2002; Wilcock et al., 2002; Reisberg et al., 2003; Tariot et al., 2004; Gauthier et al., 2005; Reisberg et al., 2006). Memantine’s mechanism of action can be explained by the fact that it is more potent and slightly less voltage-dependent than magnesium, and thus may serve as a more effective surrogate for magnesium ions (Parsons et al., 1993; Danysz and Parsons, 2003).
 
Memantine can effectively block the tonic pathological activation of NMDA receptors induced by increased presence of glutamate and/or modification of the NMDA receptor activity by the presence of amyloid protein or chronic neuroinflammation (Miguel-Hidalgo, et al.,. 2002; Wenk et al.,2006).
 
Could there be any benefits to use both cannabinoid agonists and Memantine? Figure 1 posits a simple hypothesis that might explain how the combination of these two pharmacological agents could synergistically reduce brain inflammation in AD.
 
The ability of cannabinoid agonist to reduce intracellular calcium entry (Mackie et Hille 1992) and also glutamate release (Wang, 2003) and thus avoid excessive calcium influx in the pre- and post-synaptic sides of the synapse could be beneficial, in addition to the regulatory function of memantine on the post-synaptic influx of calcium ions through the NMDA channel. The enhanced control over glutamatergic synaptic function might not be the only outcome of this combined treatment.
 
We have previously demonstrated that both WIN-55,212-2 and memantine independently have anti-inflammatory properties in a model of chronic neuroinflammation (Rosi et al., 2006, Marchalant et al., 2007), suggesting that a dual therapy in AD may also be beneficial on the inflammatory side of the disease. Moreover, both ECr stimulation and memantine have a positive effect on neurogenesis (Jin et al., 2006; Aguado et al., 2005; Galve-Roperh et al., 2007 for review) that may contribute to normal hippocampal function.
 
Figure 1
Figure 1
Schematic of the action of cannabinoids agonists and memantine at the glutamatergic synapse. ➊Memantine has the property to decrease the noise to signal ratio in presence of elevated extracellular glutamate and can regulate the influx of calcium (more ...)
 
Overall, a multi-drug approach for AD seems to emerge as a potential alternative as none of the available drug therapies are capable of altering the progression of the disease. A multimodal treatment capable of limiting inflammatory processes may lead to a slowing of the disease progression, and may also reduce some of the cognitive symptoms and thus burden on families and care-givers.
 
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Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation

Neuroscience. Author manuscript; available in PMC 2008 February 23.
Published in final edited form as:
PMCID: PMC1852513
NIHMSID: NIHMS18229
Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation
 
Yannick Marchalant, Susanna Rosi, and Gary L Wenk
Arizona Research Laboratories, Division of Neural Systems Memory and Aging, University of Arizona, Tucson, AZ (Y.M., S.R.); Neurosurgery, Physical Therapy & Rehabilitation Science, University of California at San Francisco, San Francisco, CA (S.R.); Department of psychology, Ohio State University, Columbus, OH (Y.M., G.L.W.).
 
 

Abstract
Cannabinoid receptors (CBr) stimulation induces numerous central and peripheral effects. A growing interest in the beneficial properties of manipulating the endocannabinoid system has lead to the possible involvement of CBr in the control of brain inflammation. In the present study we examined the effect of the CBr agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2), on microglial activation and spatial memory performance, using a well characterized animal model of chronic brain inflammation produced by the infusion of lipopolysaccharide (LPS, 250 ng/hr for 3 weeks) into the 4th ventricle of young rats. WIN-55212-2 (0.5 or 1.0 mg/kg/day, i.p.) was administered for three weeks. During the third week of treatment, spatial memory ability was examined using the Morris water-maze task.
 
We found that 0.5 and 1 mg/kg WIN-55212-2 reduced the number of LPS-activated microglia, while 1 mg/kg WIN-55212-2 potentiated the LPS-induced impairment of performance in the watermaze task. CB1 receptors were not expressed by microglia and astrocytes, suggesting an indirect effect of WIN on microglia activation and memory impairment. Our results emphasize the potential use of CBr agonists in the regulation of inflammatory processes within the brain; this knowledge may lead to the use of CBr agonists in the treatment of neurodegenerative diseases associated with chronic neuroinflammation, such as Alzheimer disease.


 
Microglial cells play a pivotal role as immune effectors in the central nervous system and may participate in the initiation and progression of neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease and multiple sclerosis by releasing harmful molecules such as pro-inflammatory cytokines, reactive oxygen species or complement proteins (Akiyama et al., 2000; Kim and de Vellis, 2005). Many of the pathological, immunological, biochemical and behavioral changes seen in these and other neurodegenerative diseases can be reproduced in young rats by chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999). Chronic infusion of LPS results in the activation of microglia within hippocampus and entorhinal cortex, brain regions involved in learning and memory formation (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2004).
 
Chronic brain inflammation is associated with impaired spatial memory, impaired induction of long-term potentiation, a loss of N-methyl-d-aspartate (NMDA) receptors, astrocytosis, elevated cytokines and related pro-inflammatory proteins and transcription factors (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004).
 
The endocannabinoid system may regulate many aspects of the brain’s inflammatory response, including the release of pro-inflammatory cytokines and modulation of microglial activation (Neumann, 2001; Klein, 2005).
 
The endocannabinoid system is comprised of two G-protein-coupled receptors designated as CB1 and CB2, although not all endocannabinoid effects can be explained only by these two receptors (Begg et al., 2005). CB1 receptors are expressed in the brain and are responsible for most of the behavioral effects of the cannabinoids. CB2 receptors are expressed by immune and hematopoietic cells peripherally (Begg et al., 2005), and seem to be expressed on neurons in the brainstem and the brain (Van Sickle et al., 2005; Gong et al., 2006; Onaivi et al. 2006) although their presence in the brain is controversial (Munro et al., 1993).
 
Two endogenous ligands for these receptors, arachydonylethanolamine and 2-arachidonoylglycerol (Stella, 2004), influence immune responses by inhibiting cytokine release and other anti-inflammatory actions (Klein et al., 2003; Klein, 2005).
 
Microglia also express CBr and release cytokines in response to exposure to LPS or beta-amyloid protein; this effect can be inhibited by prior cannabinoid treatment (Facchinetti et al., 2003; Ramirez et al., 2005; Sheng et al., 2005).
 
Astrocytes may also synthesize and release endocannabinoids (Walter et al., 2002). CB1 receptors have been widely studied because of their role in the psychoactive effects of the cannabis sativa plant (Δ9-etrahydrocannabinol or Δ9-THC). Δ9-THC can impair performance in rats, mice or monkeys under multiple experimental conditions (Castellano et al., 2003). Therefore, in the current study, we investigated the effect of a CBr agonist on microglial activation and spatial memory in a rodent model of chronic brain inflammation induced by LPS infusion into the 4th ventricle.
 
We used the number of immunoreactive microglia (activated) as a biomarker of brain inflammation (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004, Rosi et al., 2005) to evaluate the potential anti-inflammatory properties of the WIN 55-212-2 compound.

EXPERIMENTAL PROCEDURES
 
Subjects and surgical procedures
 
Fifty-four young (3 months old) male F-344 rats (Harlan Sprague-Dawley, Indianapolis, IN) were singly housed in Plexiglas cages with free access to food and water. The rats were maintained on a 12/12-h light-dark cycle in a temperature-controlled room (22°C) with lights off at 0800. All rats were given health checks, handled upon arrival and allowed at least one week to adapt to their new environment prior to surgery. Artificial cerebrospinal fluid (aCSF n=26) or LPS (Sigma, St-Louis, MO, E. coli, serotype 055:B5, TCA extraction, 1.0 mg/ml dissolved in aCSF, n=28) were chronically infused for 21 days through a cannula implanted into the 4th ventricle that was attached (via Tygon tubing, 0.06 O.D., and an osmotic pump connect, Model 3280P, Plastics One, Roanoke, VA) to an osmotic minipump (Alzet, Cupertino, CA, model #2004, to deliver 0.25 μl/h, Hauss-Wegrzyniak et al., 1998).
 
The aCSF vehicle contained (in mM) 140 NaCl; 3.0 KCl; 2.5 CaCl2; 1.0 MgCl2; 1.2 Na2HPO4, adjusted to pH 7.4. Rats infused with either aCSF or LPS were also administered daily the synthetic cannabinoid CB1/CB2 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2, Sigma, St-Louis, MO, 0.5 or 1.0 mg/kg, i.p.) or vehicle (Dimethylsulfoxide (100%), DMSO, Sigma, St-Louis, MO). Rats were assigned to one of the following six groups: aCSF + vehicle, LPS+ vehicle, aCSF + WIN (0.5 mg/kg), LPS + WIN (0.5mg/kg), aCSF + WIN (1 mg/kg), LPS + WIN (1 mg/kg). Drug or vehicle administration began the first day after surgery and continued throughout the behavioral testing, which began on day 14 of the LPS or aCSF infusion.
 
Behavioral Testing
 
Spatial learning ability was assessed using a 185 cm diameter water maze with white walls. The water was maintained at 26-28°C and made opaque by adding white, non-toxic, paint. The pool was in the center of a 2.3 x 2.73 x 2.5m room with multiple visual stimuli on the wall as distal cues, and a chair and a metal board against the wall of the pool as proximal cues. The circular escape platform was 11.5 cm in diameter.
 
For the spatial (hidden-platform) version of the water task, a circular escape platform was present in a constant location, submerged about 2.5 cm below the water surface. The rats were tracked by an overhead video camera connected to a VP114 tracking unit (HVS Image, England). Custom software was used to store and analyze each rat’s latency to find the submerged platform during each trial.
 
Each rat performed three training blocks per day (two training trials per block) for 4 days (24 trials total), with a 60-min inter-block interval. On each trial, the rat was released into the water from one of seven locations spaced evenly at the side of the pool, which varied randomly from trial to trial. After the rat found the escape platform or swam for a maximum of 60 sec, it was allowed to remain on the platform for 30 sec.
 
To control for possible drug-induced deficits in visual acuity and swimming ability, the same rats were also tested on a second version of this task. In this version, a visible platform raised 2 cm above the surface of the water was moved randomly to one of four locations in the tank after each trial. A total of 4 visible-platform trials were performed. Drug administration was performed 20 minutes prior to the behavioral testing. The results, i.e. latency (sec) to find the hidden platform, were analyzed by ANOVA followed by post hoc comparisons according to the method of Bonferonni/Dunn.
 
Histological procedures
 
After behavioral testing was completed, each rat was deeply anesthetized with isoflurane and prepared for a transcardiac perfusion of the brain with cold saline containing 1 U/ml heparin, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were then removed and the placement of the cannula in the 4th ventricle was confirmed. The brains were then post-fixed one hour in the same fixative and then stored (4°C) in phosphate buffer saline (PBS), pH 7.4. Free-obtained using a vibratome from perfused tissues for staining with standard avidin/biotin peroxydase or fluorescence labeling methods.
 
The monoclonal antibody OX-6 (final dilution 1:400, Pharmigen, San Diego, CA) was used to visualize activated microglia cells. This antibody is directed against Class II major histocompatibility complex (MHC II) antigen. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were incubated (4°C) overnight with primary antibodies directed against the specific epitope (MCH II).
 
Thereafter, the sections were incubated for 2h (22°C) with the secondary monoclonal antibody, rat adsorbed biotinylated horse anti-mouse immunoglobulin G (final dilution 1:200, Vector, Burlingame, CA). Sections were than incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain,
 
Elite ABC kit, Vector, Burlingame, CA). After washing again in PBS, the sections were incubated with 0.05% 3, 3’-diaminobenzidine tetrahydrochloride (Vector, Burlingame, CA) as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried and coverslipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium. The location of immunohistochemically-defined cells was examined by light microscopy.
 
Quantification of cell density in the reconstructed hippocampal coronal sections was assessed with MetaMorph imaging software (Universal Image Corporation, West Chester, PA). Briefly, areas of interest were determined as previously reported in detail (Rosi et al., 2005), their surface measured, and the immunoreactive cells numerated, allowing use to determine a number of immunoreactive cells by millimeter square in the areas of interest.
 
A polyclonal antibody directed against the first ninety-nine amino-acid residue from the human CB1 (final dilution 1:500, Affinity Bioreagents, Golden, CO) was used to visualize CB1 receptors. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were rinsed in 0.1 M TRIS buffer (TB), pH 7.4, for 15min, in 0.1 M Tris buffered saline (TBS), pH 7.4, for 15 min, blocked using 2% avidin in TBS for 30 min, rinsed in TBS for 30 min, blocked using 2% biotin for 30min, and finally rinsed in TBS for 30 min.
 
The tissue sections were then incubated in the anti-CB1 (diluted 1:500) overnight at 4°C. The antibody was diluted in a solution containing 0.1% Triton X-100 and 1% NGS in 0.1 M TBS. These sections were then rinsed in TBS for 60 min and incubated in biotinylated goat anti-rabbit IgG (diluted 1:200) for 90 min at room temperature. The sections were rinsed with TBS for 60 min and incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain, Elite ABC kit, Vector, Burlingame, CA). The sections were then rinsed with TBS for 30 min and then incubated with 0.05% 3,3’-diaminobenzidine tetrahydrochloride as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried for 24 h.Counter staining (Cresyl Violet) was performed before slides were cover-slipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium.
 
Double immunofluorescence staining
 
Free floating sections were mounted on slides and air-dried under a hound. The tissues were than processed as described in details in Rosi et al., 2005. After washing in TBS solution the polyclonal rabbit anti-CB1 (Affinity Bioreagents, Golden, CO, dilution 1:500) was apply. After 48 h of incubation at 4°C, the sections were incubated for 2 h at room temperature with the secondary anti-rabbit biotinylated antibody (Vector, Burlingame, CA), followed by incubation with Avidin+Biotin amplification system (Vector, Burlingame, CA) for 45 minutes.
 
The staining was visualized using the TSA fluorescence system CY3 (PerkinElmer Life Sciences, Emeryville, CA). After washing in TBS solution, the tissue was quenched and blocked again and incubated with the monoclonal antibody OX-6 (Pharmigen, San Diego, CA, final dilution 1:400) for 24 h or with the monoclonal antibody anti-GFAP (Chemicon, Temecula, CA, final dilution 1:500) for 24h. Before applying the biotinylated monoclonal secondary rat adsorbed antibody (Vector, Burlingame, CA) for 2 h, the tissue was incubated with Avidin Biotin Blocking Kit (Vector, Burlingame, CA) for 30 min to block cross reaction with the primary staining. Following treatment with an Avidin+Biotin amplification system (Vector, Burlingame, CA), the staining was visualized with TSA fluorescence system CY5 (PerkinElmer Life Sciences, Emeryville, CA) and the nuclei were counterstained with Sytox-Green (Molecular Probes, Eugene, OR). No staining was detected in the absence of the primary or secondary antibodies.

RESULTS
Chronic infusion of LPS and WIN-55212-2 injections were well tolerated by all rats: they gained weight normally for the duration of the study.
 
Behavior
 
No significant difference in locomotor activity (swim speed) was found across groups, p>0.1. An ANOVA performed on the latency results obtained in the water maze task revealed an overall main effect of testing day (F5, 218=16.057, p<0.0001) for all groups (See Figure 1) and an overall group effect (LPS versus aCSF) upon latency for each day of testing (F1, 328=11.367, p=0.0008 for day1; F1, 328=85.681, p<0.0001 for day2; F1,328=109.756, p<0.0001 for day3; F1, 328=96.25, p<0.0001 for day 4).
 
There was no significant effect of treatment except for day 3 (F2, 328=5.788, p=0.0034). There was a significant interaction between group and drug on days 2 and 3 (F2, 328=3.982, p=0.0196 for day 2; F2, 328=12.641, p<0.0001 for day 3). Post-hoc analyses of each testing day revealed a significant treatment effect on day 1, where LPS+WIN 1 rats were significantly impaired compared to the aCSF+WIN 1 rats (p=0.0015). On days 2 and 3, all LPS-infused rats were significantly (p<0.0033) impaired, as compared to their respective control groups. On day 4, all LPS-infused rats were significantly (p<0.0033) impaired as compared to their respective aCSF controls. There was a significant interaction between treatment and LPS-infused animals (F2, 113=5.026, p=0.081).
 
Post-hoc analyses of each testing day revealed a significant treatment effect on days 2 and 3; performance of LPS+WIN 1 rats was significantly (p<0.0033) worse as compared to the LPS+vehicle rats on those both days. Overall, the drug treatment (0.5 or 1 mg/kg) did not significantly impair performance of aCSF-infused rats and did not attenuate the impairment induced by the LPS infusion. The 1 mg/kg treatment, that did not cause any impairment in aCSF-infused rats, but did worsen the impairment observed in LPS-infused rats, demonstrating an interaction between inflammation and the highest dose of the drug used.
 
Figure 1
Figure 1
Water maze performance. On days 2 and 3, all LPS-infused animals (closed triangles, squares and circles) were significantly impaired (*p<0.0033, Bonferroni/Dunn post hoc test) compared to their control groups. On days 2 and 3, LPS+WIN 1 rats were (more ...)
Histology
Immunostaining for OX-6 (Figure 2A) revealed numerous highly activated microglia cells distributed throughout the hippocampus of LPS-infused rats (Figure 2A d). The activated microglia had a characteristic bushy morphology with increased cell body size and contracted and ramified processes (Figure 2A d). Rats infused with aCSF had very few mildly activated microglia evenly scattered throughout the brain, consistent with results from previous studies (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2005). No difference in staining was evident between the aCSF group and the aCSF rats injected with either dose of WIN (Figure 2A a-c). In LPS-infused rats that were also treated with WIN (Figure 2A e-f) the OX-6 antibody stained fewer activated microglial cells.
 
Figure 2
Figure 2
(A.) Activated microglia in the dentate gyrus. Note the diminution of activated microglia cells in the dentate gyrus of animals treated with either doses of WIN 55212-2 (e,f) compare to the LPS+vehicle group (d) (B.) Density of activated microglial cells (more ...)
 
Activated microglia cell counts
 
The number of activated microglia per millimeter square was determined in 4 different brain regions: dentate gyrus (DG), CA3 and CA1 regions of the hippocampus and the entorhinal cortex (EC) (Figure 2B). These brain regions were examined for their known implication in spatial learning (Nadel and Land, 2000). An ANOVA of the data revealed an overall main region effect (F5,210=16.057, p<0.0001) for all groups and an overall main effect of the infusion of LPS in each region examined (F5,48=32.557, p<0.0001 for DG; F5,48=23.552, p<0.0001 for CA3; F5,48=3.828, p=0.0053 for CA1;F5,48=19.308, p<0.0001 for EC).
 
In the DG, CA3 and EC, all LPS infused rats had a significantly (p<0.0033) higher density of activated microglia compared to their respective control groups, consistent with our previous results (Rosi et al., 2005). In the DG and CA3 the number of activated microglia was significantly (p<0.0033) reduced in LPS-infused rats given either dose of WIN, as compared to rats in the LPS+vehicle group (DG, 41.4% reduction for 0.5mg/kg and 40.6% reduction for 1mg/kg; CA3, 43.7% reduction for WIN 0.5mg/kg and 49.4% reduction for WIN 1mg/kg). In the EC, despite a 24.2% reduction for WIN 0.5mg/kg and 32.4% reduction for WIN 1mg/kg, no significant difference was found between the LPS-infused groups. In the CA1 region of the hippocampus, there was a significant (p<0.0033) difference only between the aCSF and LPS+vehicle groups. Overall, the effects of WIN were not dose-dependent in the DG and CA3 and not significant within the CA1 or EC brain regions.
 
CB1 receptors
 
The distribution of the CB1 receptors observed following our staining is in accordance with previous studies (Tsou et al., 1998) (Figure 3a). Neuronal CB1 immunoreactivity was found in the hippocampus, striatum, amygdala as well as in the somatosensory, cingulate and entorhinal cortices. The apparent density of immunoreactive cells in the areas of interest (DG, CA3, CA1, or EC) did not vary across groups.
 
Figure 3
 
Immunoreactivity (IR) of CBr in the CA3 region of the hippocampus. (a) CB1-IR (DAB stain) is seen in the cytoplasm of all CA3 neuronal cell bodies (counterstaining with cresyl violet). (b) CB1-IR (red) and MCH-II-IR (green) do no co-localize, as indicated (more ...)
No co-localization between CB1 receptors and activated microglial cells
Double-immunofluorescence staining for CB1 and activated microglial cells performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3b). These results indicate that CB1 receptors are not present on activated microglial cells in response to LPS infusion or treatment with a CBr agonist.
 
No co-localization between CB1 and astrocytes
 
Double-immunofluorescence staining for CB1 and astrocytes performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3c). These results indicate that CB1 are not present on astrocytes in response to LPS infusion or treatment with a CBr agonist.

DISCUSSION
 
The results demonstrate that a CB1/CB2 agonist, WIN-55212-2, prevents microglial cell activation during LPS-induced chronic neuroinflammation in young rats. The effects of this agonist were not dependent on direct CB1 receptors stimulation of microglia or astrocytes, were region dependant and did not reverse the LPS-induced impairment in a spatial memory task.
 
The neurodegeneration associated with AD may result from prolonged activation of microglia and a chronic elevation of cytokines and nitric oxide (Akiyama et al., 2000; Streit, 2004) leading to a cascade of self-propagating cellular events that alters the expression of cannabinoid receptors (Minghetti and Levi, 1998; Bernardino et al., 2005; Klein, 2005). Endocannabinoids are implicated in the modulation of the central inflammatory response by neurons and glia (Klein et al., 2003; Klein, 2005) and may have a neuroprotective role in several neuroinflammation related diseases (Klein, 2005). Cannabinoid agonists can prevent the activation of microglia by β-amyloid, reduce the subsequent release of TNF-α(Ramirez et al., 2005), and attenuate the induction and release of nitric oxide by cultured microglia (Waksman et al., 1999).
 
An inhibition of glutamate release by cannabinoids and the subsequent reduction of the calcium influx via NMDA channels (Piomelli, 2003; Takahashi and Castillo, 2006) have also been demonstrated. Additionally, cannabinoids can attenuate oxidative stress and subsequent toxicity (Hampson and Grimaldi, 2001) and induce the expression of brain-derived neurotrophic factor following the infusion of kainic acid (Marsicano et al., 2003).
 
CBr are expressed in senile plaques (Benito et al., 2003; Ramirez et al., 2005) and the number of CB1 receptor-immunoreactive neurons is greatly reduced in areas of microglial activation in the AD brain (Ramirez et al., 2005). In contrast, we did not find evidence for changes in CB1 receptor expression on hippocampal neurons in response to brain inflammation or cannabinoid receptor stimulation. This absence of changes in CB1 receptors on neurons in our model may be related to the fact that the chronic infusion of LPS into young rats does not induce senile plaque formation (Hauss-Wegrzyniak et al., 1998) or neuronal loss in the hippocampus (Rosi et al., 2005), important factors that likely influence the expression of CBr-positive neuron in post-mortem studies on AD brains from aged humans. The LPS infusion into the 4th ventricle produces widespread activation of glia and impaired performance in the water maze task (Hauss-Wegrzyniak et al., 1998). In contrast, an infusion of amyloid into a lateral ventricle (Ramirez et al., 2005) produced a more localized and limited glial activation (frontal cortex) and performance impairment that responded positively to treatment with WIN 55-212-2 (Ramirez et al., 2005). This difference may explain why WIN 55-212-2 in our model did not reverse the impairment induced by LPS in the 4th ventricle.
 
In the current study, CB1 receptors were not co-localized with MCH II-positive microglia or GFAP-positive astrocytes within the hippocampus; the presence of these receptors appeared to be solely neuronal (Figure 3). This suggests an indirect role of CBr upon microglia that is linked to the modulation of neuronal activity by stimulation of the endocannabinoid system. Our results are consistent with previous in vivo findings (Tsou et al., 1998) that describe CBr receptors located on hippocampal neurons, and their modulator role upon glutamatergic and GABAergic function (Takahashi and Castillo, 2006; Katona et al., 1999). CBr are present on astrocytes and microglial cells taken from humans, monkeys, rats and mice when studied in vitro (Stella 2004).
 
The contradictory findings we report may be related to many factors, such as the different species that have been studied, the microenvironment of the cells (in vivo vs in vitro), antibody sensitivity and selectivity, and the age or pathological condition under examination. Further experiments must be performed to determine clearly the characteristic and changes of the endocannabinoid system in our model and thus explain our present findings.
 
We have previously speculated that LPS induces a cascade of inflammatory processes that leads to an elevation in extracellular glutamate and activation of NMDA receptors (Wenk et al., 2006). The selective antagonism of NMDA receptors reduced microglia activation (Rosi et al., 2006) similar to that reported in the current study using WIN-55212-2. Taken together these findings are consistent with the hypothesis that the ability of endocannabinoids to reduce the release of glutamate within the hippocampus underlies the reduction of microglia activation by WIN-55212-2.
 
Interestingly, our results demonstrated an interaction between the presence of brain inflammation and the actions of the cannabinoid agonist at a dose (1 mg/kg/day i.p. of WIN 55-212-2) that did not impair performance in the control rats. Surprisingly, those doses of agonist (0.5 and 1 mg/kg/day i.p. of WIN 55-212-2) also did not improve performance, as might be expected given the results of our previous investigations using more typical anti-inflammatory drugs (Hauss-Wegrzyniak et al., 1999).
 
We speculate that this was because the WIN 55-212-2 treatment was not able to reverse totally the activation of microglia that had been induced by the chronic LPS infusion; in the current study, the reduction in the number of activated microglia was only about 40-50%, in contrast to the ability of an non-steroidal anti-inflammatory drug’s ability to reduce the number of activated microglia almost completely.
 
This partial reduction in microglia activation might thus not be sufficient to reverse the spatial memory impairment produced by the LPS infusion. This result is of particular importance with regard to patients suffering with a disease associated with brain inflammation, e.g. AD, Parkinson’s disease or multiple sclerosis, who are also using marijuana.
The current report is the first to our knowledge to demonstrate the modulatory role of cannabinoids in an animal model of chronic neuroinflammation, pointing out the effectiveness of a CBr agonist on the consequences of LPS mediated neuroinflammation at a dose (0.5 mg/kg/day i.p. of WIN-55212-2) that does not impair performance in a patial memory task.
 
These results further advocate for the manipulation of the endocannabinoid system to diminish the consequences of neuroinflammation in progression of AD and others inflammation-related diseases (Klein, 2005).
 
Acknowledgments
 
We thank A. Sy, P. Raniero, F. Cerbai for technical assistance and Dr V. Ramirez-Amaya for critical experimental procedure assistance.
List of abbreviations
 
Δ9-THCΔ9-tetrahydrocannabinol
 
aCSFartificial cerebral spinal fluid
 
ADAlzheimer’s disease
 
CB1cannabinoid receptor 1
 
CB2cannabinoid receptor 2
 
CBrcannabinoid receptors
 
DGDentate gyrus
 
ECEntorhinal cortex
 
LPSLipopolysaccharide
 
NMDAN-methyl-d-aspartate
 
PBSphosphate buffer saline
 
TBSTris buffer saline
 
WIN-55212-2(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate

 
Financial support:This study has been supported by AG10546 (GLW).
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Marijuana reduces memory impairment

The more research they do, the more evidence Ohio State University scientists find that specific elements of marijuana can be good for the aging brain by reducing inflammation there and possibly even stimulating the formation of new brain cells.

 

The research suggests that the development of a legal drug that contains certain properties similar to those in marijuana might help prevent or delay the onset of Alzheimer's disease. Though the exact cause of Alzheimer's remains unknown, chronic inflammation in the brain is believed to contribute to memory impairment.


Any new drug's properties would resemble those of tetrahydrocannabinol, or THC, the main psychoactive substance in the cannabis plant, but would not share its high-producing effects. THC joins nicotine, alcohol and caffeine as agents that, in moderation, have shown some protection against inflammation in the brain that might translate to better memory late in life.

"It's not that everything immoral is good for the brain. It's just that there are some substances that millions of people for thousands of years have used in billions of doses, and we're noticing there's a little signal above all the noise," said Gary Wenk, professor of psychology at Ohio State and principal investigator on the research.

Wenk's work has already shown that a THC-like synthetic drug can improve memory in animals. Now his team is trying to find out exactly how it works in the brain.


The most recent research on rats indicates that at least three receptors in the brain are activated by the synthetic drug, which is similar to marijuana. These receptors are proteins within the brain's endocannabinoid system, which is involved in memory as well as physiological processes associated with appetite, mood and pain response.

This research is also showing that receptors in this system can influence brain inflammation and the production of new neurons, or brain cells.

"When we're young, we reproduce neurons and our memory works fine. When we age, the process slows down, so we have a decrease in new cell formation in normal aging. You need those cells to come back and help form new memories, and we found that this THC-like agent can influence creation of those cells," said Yannick Marchalant, a study coauthor and research assistant professor of psychology at Ohio State.

Marchalant described the research in a poster presentation Wednesday (11/19) at the Society for Neuroscience meeting in Washington, D.C.

Knowing exactly how any of these compounds work in the brain can make it easier for drug designers to target specific systems with agents that will offer the most effective anti-aging benefits, said Wenk, who is also a professor of neuroscience and molecular virology, immunology and medical genetics.

"Could people smoke marijuana to prevent Alzheimer's disease if the disease is in their family? We're not saying that, but it might actually work. What we are saying is it appears that a safe, legal substance that mimics those important properties of marijuana can work on receptors in the brain to prevent memory impairments in aging. So that's really hopeful," Wenk said.

One thing is clear from the studies: Once memory impairment is evident, the treatment is not effective. Reducing inflammation and preserving or generating neurons must occur before the memory loss is obvious, Wenk said.


Marchalant led a study on old rats using the synthetic drug, called WIN-55212-2 (WIN), which is not used in humans because of its high potency to induce psychoactive effects.


The researchers used a pump under the skin to give the rats a constant dose of WIN for three weeks – a dose low enough to induce no psychoactive effects on the animals. A control group of rats received no intervention. In follow-up memory tests, in which rats were placed in a small swimming pool to determine how well they use visual cues to find a platform hidden under the surface of the water, the treated rats did better than the control rats in learning and remembering how to find the hidden platform.


"Old rats are not very good at that task. They can learn, but it takes them more time to find the platform. When we gave them the drug, it made them a little better at that task," Marchalant said.


In some rats, Marchalant combined the WIN with compounds that are known to block specific receptors, which then offers hints at which receptors WIN is activating. The results indicated the WIN lowered the rats' brain inflammation in the hippocampus by acting on what is called the TRPV1 receptor. The hippocampus is responsible for short-term memory.

With the same intervention technique, the researchers also determined that WIN acts on receptors known as CB1 and CB2, leading to the generation of new brain cells – a process known as neurogenesis. Those results led the scientists to speculate that the combination of lowered inflammation and neurogenesis is the reason the rats' memory improved after treatment with WIN.


The researchers are continuing to study the endocannabinoid system's role in regulating inflammation and neuron development. They are trying to zero in on the receptors that must be activated to produce the most benefits from any newly developed drug.

What they already know is THC alone isn't the answer.


"The end goal is not to recommend the use of THC in humans to reduce Alzheimer's," Marchalant said. "We need to find exactly which receptors are most crucial, and ideally lead to the development of drugs that specifically activate those receptors. We hope a compound can be found that can target both inflammation and neurogenesis, which would be the most efficient way to produce the best effects."

The National Institutes of Health supported this work.

 

 

 

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The activation of cannabinoid CB2 receptors stimulates in situ and in vitro beta-amyloid removal by human macrophages.

Tolón RM, Núñez E, Pazos MR, Benito C, Castillo AI, Martínez-Orgado JA, Romero J.

Laboratorio de Apoyo a la Investigación, Hospital Universitario Fundación Alcorcón and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, 28922 Alcorcón, Madrid, Spain.

 

Abstract

 

The endocannabinoid system is a promising therapeutic target in a wide variety of diseases. However, the non-desirable psychotropic effects of natural and synthetic cannabinoids have largely counteracted their clinical usefulness. These effects are mostly mediated by cannabinoid receptors of the CB(1) type, that exhibit a wide distribution in neuronal elements of the CNS.

 

Thus, the presence of other elements of this system in the CNS, such as CB(2) receptors, may open new possibilities for the development of cannabinoid-based therapies. These receptors are almost absent from the CNS in normal conditions but are up-regulated in glial cells under chronic neuroinflammatory stimuli, as has been described in Alzheimer's disease.

 

To understand the functional role of these receptors, we tested their role in the process of beta-amyloid removal, that is currently considered as one of the most promising experimental approaches for the treatment of this disease. Our results show that a CB(2) agonist (JWH-015) is capable of inducing the removal of native beta-amyloid removal from human frozen tissue sections as well as of synthetic pathogenic peptide by a human macrophage cell line (THP-1).

 

Remarkably, this effect was achieved at low doses (maximum effect at 10 nM) and was specific for this type of cells, as U373MG astrocytoma cells did not respond to the treatment. The effect was CB(2)-mediated, at least partially, as the selective CB(2) antagonist SR144528 prevented the JWH-015-induced plaque removal in situ. These data corroborate the possible therapeutic interest of CB(2) cannabinoid specific chemicals in the treatment of Alzheimer's disease.

 

PMID: 19505450 [PubMed - indexed for MEDLINE]

 

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Attacking Alzheimer's with Red Wine and Marijuana


Two new studies point to a wonderful way to ward off Alzheimer's disease and other forms of age-related memory los

 

This article first appeared on Miller-McCune.com.

 

Two new studies suggest that substances usually associated with dulling the mind -- marijuana and red wine -- may help ward off Alzheimer's disease and other forms of age-related memory loss. Their addition comes as another study dethrones folk remedy ginkgo biloba as proof against the disease.

 

At a November meeting of the Society of Neuroscience in Washington, D.C., researchers from Ohio State University reported that THC, the main psychoactive substance in the cannabis plant, may reduce inflammation in the brain and even stimulate the formation of new brain cells.

 

Meanwhile, in the Nov. 21 issue of the Journal of Biological Chemistry, neurologist David Teplow of the University of California, Los Angeles reported that polyphenols -- naturally occurring components of red wine -- block the formation of proteins that build the toxic plaques thought to destroy brain cells. In addition, these substances can reduce the toxicity of existing plaques, thus reducing cognitive deterioration.

 

Together, the studies suggest scientists are gaining a clearer understanding of the mechanics of memory deterioration and discovering some promising approaches to prevention.

 

Previous research has suggested that polyphenols -- which are found in high concentrations in tea, nuts and berries, as well as cabernets and merlots -- may inhibit or prevent the buildup of toxic fibers in the brain. These fibers, which are primarily composed of two specific proteins, form the plaques that have long been associated with Alzheimer's disease.

 

UCLA's Teplow and his colleagues monitored how these proteins folded up and stuck to each other to produce aggregates that killed nerve cells in mice. They then treated the proteins with a polyphenol compound extracted from grape seeds. They discovered the polyphenols blocked the formation of the toxic aggregates.

 

"What we found is pretty straightforward," Teplow declared. "If the amyloid beta proteins can't assemble, toxic aggregates can't form, and, thus, there is no toxicity." If this also proves true in human brains, it means administration of the compound to Alzheimer's patients could "prevent disease development and also ameliorate existing disease," he said. Human clinical trials are upcoming.

 

At Ohio State, researchers led by psychologist Gary Wenk are studying the protective effects of tetrahydrocannabinol, commonly known as THC. They found that administering a THC-like synthetic drug to older rats performed better at a memory test than a control group of non-medicated elderly rodents.

 

In some of the rats, the drug apparently lowered inflammation in the hippocampus -- the region of the brain responsible for short-term memory. It also seems to have stimulated the generation of new brain cells.

 

"When we're young, we reproduce neurons and our memory works fine," said co-author Yannick Marchalant, another Ohio State psychologist. "When we age, the process slows down, so we have a decrease in new cell formation in normal aging. You need those cells to come back and help form new memories, and we found that this THC-like agent can influence creation of those cells."

 

Wenk added two cautionary notes to his report. First, to be effective, any such treatment along these lines would have to take place before memory loss is obvious. Second, the researchers still have much work to do.

 

"We need to find exactly which receptors are most crucial" to the generation of new brain cells, he said. This discovery would "ideally lead to the development of drugs that specifically activate those receptors."

 

In the meantime, should aging baby boomers who are worried about old-age mental impairment light up a joint? Wenk was cautious in his answer, no doubt because marijuana is suspected to be harmful to health in other ways.



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Pot joins the fight against Alzheimer's, memory loss

 

A large-scale study released this week showed that the herb gingko biloba has no effect in preventing dementia or Alzheimer’s disease. But alternative medicine aficionados may find hope in a new research touting the bennies of another "herb" in preserving memory.



Scientists from Ohio State University report that marijuana, contrary to the conventional wisdom,  may help ward off Alzheimer's and keep recall sharp. Their findings, released today at the Society for Neuroscience meeting in Washington D.C.:  chemical components  of marijuana reduce inflammation and stimulate the production of new brain cells, thereby enhancing memory.



The team suggested that a  drug could be formulated that would resemble tetrahydroannibol, or THC, the psychoactive  ingredient in pot sans making the user high. But the research  may ultimately drive those who fear impending dementia to roll their own solution to the problem.



Study co-author Gary Wenk, a professor of psychology, had already devised a preliminary version of a THC-like synthetic drug that improves memory in lab animals. His team at the meeting said that it works by activating at least three receptors in the brain targeted by THC—proteins on the surface of nerve cells  that then trigger cellular processes resulting in reduced inflammation and production of new brain cells that can boost recall. Understanding how the compounds work  may pave the way for a pharmaceutical company to prepare its own  med for human clinical trials.



The researchers ducked the obvious question of whether it might be simpler, faster and cheaper to simply light up a joint. “Could people smoke marijuana to prevent Alzheimer’s disease if the disease is in their family?" Wenk said in a statement. "We’re not saying that, but it might actually work.”

 


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