Phytocannabinoids and Cannabis-Based Products as Alternative Pharmacotherapy in Neurodegenerative Diseases: From Hypothesis to Clinical Practice.

Historically, Cannabis is one of the first plants to be domesticated and used in medicine, though only in the last years the amount of Cannabis-based products or medicines has increased worldwide. Previous preclinical studies and few published clinical trials have demonstrated the efficacy and safety of Cannabis-based medicines in humans. Indeed, Cannabis-related medicines are used to treat multiple pathological conditions, including neurodegenerative disorders. In clinical practice, Cannabis products have already been introduced to treatment regimens of Alzheimer's disease, Parkinson's disease and Multiple Sclerosis's patients, and the mechanisms of action behind the reported improvement in the clinical outcome and disease progression are associated with their anti-inflammatory, immunosuppressive, antioxidant, and neuroprotective properties, due to the modulation of the endocannabinoid system. In this review, we describe the role played by the endocannabinoid system in the physiopathology of Alzheimer, Parkinson, and Multiple Sclerosis, mainly at the neuroimmunological level. We also discuss the evidence for the correlation between phytocannabinoids and their therapeutic effects in these disorders, thus describing the main clinical studies carried out so far on the therapeutic performance of Cannabis-based medicines.

Quality of Life and a Surveillant Endocannabinoid System.

The endocannabinoid system (ECS) is an important brain modulatory network. ECS regulates brain homeostasis throughout development, from progenitor fate decision to neuro- and gliogenesis, synaptogenesis, brain plasticity and circuit repair, up to learning, memory, fear, protection, and death. It is a major player in the hypothalamic-peripheral system-adipose tissue in the regulation of food intake, energy storage, nutritional status, and adipose tissue mass, consequently affecting obesity. Loss of ECS control might affect mood disorders (anxiety, hyperactivity, psychosis, and depression), lead to drug abuse, and impact neurodegenerative (Alzheimer's, Parkinson, Huntington, Multiple, and Amyotrophic Lateral Sclerosis) and neurodevelopmental (autism spectrum) disorders. Practice of regular physical and/or mind-body mindfulness and meditative activities have been shown to modulate endocannabinoid (eCB) levels, in addition to other players as brain-derived neurotrophic factor (BDNF). ECS is involved in pain, inflammation, metabolic and cardiovascular dysfunctions, general immune responses (asthma, allergy, and arthritis) and tumor expansion, both/either in the brain and/or in the periphery. The reason for such a vast impact is the fact that arachidonic acid, a precursor of eCBs, is present in every membrane cell of the body and on demand eCBs synthesis is regulated by electrical activity and calcium shifts. Novel lipid (lipoxins and resolvins) or peptide (hemopressin) players of the ECS also operate as regulators of physiological allostasis. Indeed, the presence of cannabinoid receptors in intracellular organelles as mitochondria or lysosomes, or in nuclear targets as PPARγ might impact energy consumption, metabolism and cell death. To live a better life implies in a vigilant ECS, through healthy diet selection (based on a balanced omega-3 and -6 polyunsaturated fatty acids), weekly exercises and meditation therapy, all of which regulating eCBs levels, surrounded by a constructive social network. Cannabidiol, a diet supplement has been a major player with anti-inflammatory, anxiolytic, antidepressant, and antioxidant activities. Cognitive challenges and emotional intelligence might strengthen the ECS, which is built on a variety of synapses that modify human behavior. As therapeutically concerned, the ECS is essential for maintaining homeostasis and cannabinoids are promising tools to control innumerous targets.

Maternal omega-3 intake differentially affects the endocannabinoid system in the progeny`s neocortex and hippocampus: Impact on synaptic markers.

Omega-3 (n-3) polyunsaturated fatty acids (PUFA) and the endocannabinoid system (ECS) modulate several functions through neurodevelopment including synaptic plasticity mechanisms. The interplay between n-3PUFA and the ECS during the early stages of development, however, is not fully understood. This study investigated the effects of maternal n-3PUFA supplementation (n-3Sup) or deficiency (n-3Def) on ECS and synaptic markers in postnatal offspring. Female rats were fed with a control, n-3Def, or n-3Sup diet from 15 days before mating and during pregnancy. The cerebral cortex and hippocampus of mothers and postnatal 1-2 days offspring were analyzed. In the mothers, a n-3 deficiency reduced CB1 receptor (CB1R) protein levels in the cortex and increased CB2 receptor (CB2R) in both cortex and hippocampus. In neonates, a maternal n-3 deficiency reduced the hippocampal CB1R amount while it increased CB2R. Additionally, total GFAP isoform expression was increased in both cortex and hippocampus in neonates of the n-3Def group. Otherwise, maternal n-3 supplementation increased the levels of n-3-derived endocannabinoids, DHEA and EPEA, in the cortex and hippocampus and reduced 2-arachidonoyl-glycerol (2-AG) concentrations in the cortex of the offspring. Furthermore, maternal n-3 supplementation also increased PKA phosphorylation in the cortex and ERK phosphorylation in the hippocampus. Synaptophysin immunocontent in both regions was also increased. In vitro assays showed that the increase of synaptophysin in the n-3Sup group was independent of CB1R activation. The findings show that variations in maternal dietary omega-3 PUFA levels may impact differently on the ECS and molecular markers in the cerebral cortex and hippocampus of the progeny.

Cannabinoids Induce Cell Death and Promote P2X7 Receptor Signaling in Retinal Glial Progenitors in Culture.

Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [3H]-thymidine incorporation was observed when cultures were incubated with 0.5-1.0 µM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5-5.0 µM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50 mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1 mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.

Cellulose exopolysaccharide from sugarcane molasses as a suitable substrate for 2D and 3D neuron and astrocyte primary cultures.

Bacteria-synthesized polysaccharides have attracted interest for biomedical applications as promising biomaterials to be used as implants and scaffolds. The present study tested the hypothesis that cellulose exopolysaccharide (CEC) produced from sugarcane molasses of low cost and adequate purity would be suitable as a template for 2D and 3D neuron and/or astrocyte primary cultures, considering its low toxicity. CEC biocompatibility in these primary cultures was evaluated with respect to cell viability, adhesion, growth and cell function (calcium imaging). Polystyrene or Matrigel® matrix were used as comparative controls. We demonstrated that the properties of this CEC in the 2D or 3D configurations are suitable for differentiation of cortical astrocytes and neurons in single or mixed cultures. No toxicity was detected in neurons that showed NMDA-induced Ca2+ influx. Unlike other polysaccharides of bacterial synthesis, the CEC was efficient as a support even in the absence of surface conjugation with extracellular matrix proteins, maintaining physiological characteristics of cultured neural cells. These observations open up the perspective for development of a novel 3D biofunctional scaffold produced from bacterial cellulose and obtained from renewable sources whose residues are not pollutants. Its low cost and possibility to be manufactured in scale are also suitable for potential applications in regenerative medicine.

Low omega-6/omega-3 ratio in a maternal protein-deficient diet promotes histone-3 changes in progeny neural cells and favors leukemia inhibitory factor genetranscription.

Omega-3 (n-3) fatty acids modulate epigenetic changes critical to genesis and differentiation of neural cells. Conversely, maternal protein-malnutrition can negatively modify these changes. This study investigated whether a low n-6/n-3 ratio in a maternal diet could favor histone-3 (H3) modifications, gene transcription and differentiation in the offspring neural cells even under protein-deficiency. Female rats fed a control (Ct), or 3 types of multideficient diets differing in protein levels or linoleic/alpha-linolenic fatty acid ratios (RBD, RBD-C, RBD-SO) from 30 days prior to mating and during pregnancy. Cerebral cortex tissue and cortical cultures of progeny embryonic neurons and postnatal astrocytes were analyzed. H3K9 acetylation and H3K27 or H3K4 di-methylation levels were assessed by flow cytometry and/or immunocytochemistry. In astrocyte cultures and cortical tissue, the GFAP protein levels were assessed. Glial derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) gene expression were evaluated in the cortical tissue. GFAP levels were similar in astrocytes of Ct, RBD and RBD-C, but 65% lower in RBD-SO group. Higher levels of H3K9Ac were found in the neurons and H3K4Me2 in the astrocytes of the RBD group. No intergroup difference in the cortical GDNF mRNA expression or the H3K27Me2 levels in astrocytes was detected. LIF mRNA levels were higher in the RDB (P=.002) or RBD-C (P=.004) groups than in the control. The findings indicate the importance of dietary n-3 availability for the brain, even under a protein-deficient condition, inducing Histone modifications and increasing LIF gene transcription, involved in neural cell differentiation and reactivity.

Polyunsaturated fatty acids and endocannabinoids in health and disease.

Polyunsaturated fatty acids (PUFAs) are lipid derivatives of omega-3 (docosahexaenoic acid, DHA, and eicosapentaenoic acid, EPA) or of omega-6 (arachidonic acid, ARA) synthesized from membrane phospholipids and used as a precursor for endocannabinoids (ECs). They mediate significant effects in the fine-tune adjustment of body homeostasis. Phyto- and synthetic cannabinoids also rule the daily life of billions worldwide, as they are involved in obesity, depression and drug addiction. Consequently, there is growing interest to reveal novel active compounds in this field. Cloning of cannabinoid receptors in the 90s and the identification of the endogenous mediators arachidonylethanolamide (anandamide, AEA) and 2-arachidonyglycerol (2-AG), led to the characterization of the endocannabinoid system (ECS), together with their metabolizing enzymes and membrane transporters. Today, the ECS is known to be involved in diverse functions such as appetite control, food intake, energy balance, neuroprotection, neurodegenerative diseases, stroke, mood disorders, emesis, modulation of pain, inflammatory responses, as well as in cancer therapy. Western diet as well as restriction of micronutrients and fatty acids, such as DHA, could be related to altered production of pro-inflammatory mediators (e.g. eicosanoids) and ECs, contributing to the progression of cardiovascular diseases, diabetes, obesity, depression or impairing conditions, such as Alzheimer' s disease. Here we review how diets based in PUFAs might be linked to ECS and to the maintenance of central and peripheral metabolism, brain plasticity, memory and learning, blood flow, and genesis of neural cells.

Fighting Oxidative Stress: Increased Resistance of Male Rat Cerebellum at Weaning Induced by Low Omega 6/Omega 3 Ratio in a Protein-Deficient Diet.

The cerebellum is vulnerable to malnutrition effects. Notwithstanding, it is able to incorporate higher amount of docosahexaenoic acid (DHA) than the cerebral cortex (Cx) when low n-6/n-3 fatty acid ratio is present in a multideficient diet. Considering importance of DHA for brain redox balance, we hypothesize that this cerebellum feature improves its antioxidant status compared to the Cx. A chronic malnutrition status was induced on dams before mating and kept until weaning or adulthood (offspring). A group nutritionally rehabilitated from weaning was also analyzed. Morphometric parameters, total-superoxide dismutase (t-SOD) and catalase activities, lipoperoxidation (LP), nitric oxide (NO), reduced (GSH) and oxidized (GSSG) glutathione, reactive oxygen species (ROS), and reduced nicotinamide adenine dinucleotide/phosphate levels were assessed. Both ROS and LP levels were increased (∼53 %) in the Cx of malnourished young animals while the opposite was seen in the cerebellum (72 and 20 % of the control, respectively). Consistently, lower (∼35 %) and higher t-SOD (∼153 %) and catalase (CAT) (∼38 %) activities were respectively detected in the Cx and cerebellum compared to the control. In malnourished adult animals, redox balance was maintained in the cerebellum and recovered in the Cx (lower ROS and LP levels and higher GSH/GSSG ratio). NO production was impaired by malnutrition at either age, mainly in the cerebellum. The findings suggest that despite a multinutrient deficiency and a modified structural development, a low dietary n-6/n-3 ratio favors early antioxidant resources in the male cerebellum and indicates an important role of astrocytes in the redox balance recovery of Cx in adulthood.