Effects of Chronic, Low-Dose Cannabinoids, Cannabidiol, Delta-9-Tetrahydrocannabinol and a Combination of Both, on Amyloid Pathology in the 5xFAD Mouse Model of Alzheimer's Disease.

Background: There is an urgent need for novel therapies to treat Alzheimer's disease. Among others, the use of cannabinoids such as delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) has been proposed as a putative approach based on their anti-inflammatory effects. Methods: The present work was designed to explore the effects of chronic (28 days) treatment with low doses of cannabinoids: CBD (0.273 mg/kg), THC (0.205 mg/kg) or a combination of both (CBD:THC; 0.273 mg/kg:0.205 mg/kg) in the 5xFAD mouse model of AD. Results: Our data revealed that THC-treated 5xFAD mice (but not other treatment groups) exhibited anxiogenic and depressant-like behavior. A significant improvement in spatial memory was observed only in the CBD:THC-treated group. Interestingly, all cannabinoid-treated groups showed significantly increased cortical levels of the insoluble form of beta amyloid 1-42. These effects were not accompanied by changes in molecular parameters of inflammation at the mRNA or protein level. Conclusions: These data reveal differential effects of chronic, low-dose cannabinoids and point to a role of these cannabinoids in the processing of amyloid peptides in the brains of 5xFAD mice.

Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.

The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB1 receptor. In Alzheimer's disease (AD), amyloid beta (Aß)-containing aggregates induce a chronic inflammatory response leading to reactivity of both microglia and astrocytes. However, how this glial response impacts on the glial CB1 receptor expression in the subiculum of a mouse model of AD, a brain region particularly affected by large accumulation of plaques and concomitant subcellular changes in microglia and astrocytes, is unknown. The CB1 receptor localization in both glial cells was investigated in the subiculum of male 5xFAD/CB2 EGFP/f/f (AD model) and CB2 EGFP/f/f mice by immuno-electron microscopy. The findings revealed that glial CB1 receptors suffer remarkable changes in the AD mouse. Thus, CB1 receptor expression increases in reactive microglia in 5xFAD/CB2 EGFP/f/f , but remains constant in astrocytes with CB1 receptor labeling rising proportionally to the perimeter of the reactive astrocytes. Not least, the CB1 receptor localization in microglial processes in the subiculum of controls and closely surrounding amyloid plaques and dystrophic neurites of the AD model, supports previous suggestions of the presence of the CB1 receptor in microglia. These findings on the correlation between glial reactivity and the CB1 receptor expression in microglial cells and astrocytes, contribute to the understanding of the role of the endocannabinoid system in the pathophysiology of Alzheimer's disease.

Insulin regulates neurovascular coupling through astrocytes.

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.

Cannabinoid CB2 Receptors Modulate Microglia Function and Amyloid Dynamics in a Mouse Model of Alzheimer's Disease.

The distribution and roles of the cannabinoid CB2 receptor in the CNS are still a matter of debate. Recent data suggest that, in addition to its presence in microglial cells, the CB2 receptor may be also expressed at low levels, yet biologically relevant, in other cell types such as neurons. It is accepted that the expression of CB2 receptors in the CNS is low under physiological conditions and is significantly elevated in chronic neuroinflammatory states associated with neurodegenerative diseases such as Alzheimer's disease. By using a novel mouse model (CB2 EGFP/f/f), we studied the distribution of cannabinoid CB2 receptors in the 5xFAD mouse model of Alzheimer's disease (by generating 5xFAD/CB2 EGFP/f/f mice) and explored the roles of CB2 receptors in microglial function. We used a novel selective and brain penetrant CB2 receptor agonist (RO6866945) as well as mice lacking the CB2 receptor (5xFAD/CB2 -/-) for these studies. We found that CB2 receptors are expressed in dystrophic neurite-associated microglia and that their modulation modifies the number and activity of microglial cells as well as the metabolism of the insoluble form of the amyloid peptide. These results support microglial CB2 receptors as potential targets for the development of amyloid-modulating therapies.

Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer's disease.

BACKGROUND: The complex pathophysiology of Alzheimer's disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit. METHODS: In this work, we use electrophysiology, flow cytometry and molecular analysis to evaluate the cellular and molecular mechanisms underlying the improvement associated to the increased endocannabinoid tone in the 5xFAD mouse- model. RESULTS: We demonstrate that the chronic enhancement of the endocannabinoid tone rescues hippocampal synaptic plasticity in the 5xFAD mouse model. At the CA3-CA1 synapse, both basal synaptic transmission and long-term potentiation (LTP) of synaptic transmission are normalized upon FAAH genetic inactivation, in a CB1 receptor (CB1R)- and TRPV1 receptor-independent manner. Dendritic spine density in CA1 pyramidal neurons, which is notably decreased in 6-month-old 5xFAD animals, is also restored. Importantly, we reveal that the expression of microglial factors linked to phagocytic activity, such as TREM2 and CTSD, and other factors related to amyloid beta clearance and involved in neuron-glia crosstalk, such as complement component C3 and complement receptor C3AR, are specifically upregulated in 5xFAD/FAAH-/- animals. CONCLUSION: In summary, our findings support the therapeutic potential of modulating, rather than suppressing, neuroinflammation in Alzheimer's disease. In our model, the long-term enhancement of the endocannabinoid tone triggered augmented microglial activation and amyloid beta phagocytosis, and a consequent reversal in the neuronal phenotype associated to the disease.

Role of interleukin 1-beta in the inflammatory response in a fatty acid amide hydrolase-knockout mouse model of Alzheimer's disease.

The search for novel therapies for the treatment of Alzheimer's disease is an urgent need, due to the current paucity of available pharmacological tools and the recent failures obtained in clinical trials. Among other strategies, the modulation of amyloid-triggered neuroinflammation by the endocannabinoid system seems of relevance. Previous data indicate that the enhancement of the endocannabinoid tone through the inhibition of the enzymes responsible for the degradation of their main endogenous ligands may render beneficial effects. Based on previously reported data, in which we described a paradoxical effect of the genetic deletion of the fatty acid amide hydrolase, we here aimed to expand our knowledge on the role of the endocannabinoid system in the context of Alzheimer's disease. To that end, we inhibited the production of interleukin-1ß, one of the main inflammatory cytokines involved in the neuroinflammation triggered by amyloid peptides, in a transgenic mouse model of this disease by using minocycline, a drug known to impair the synthesis of this cytokine. Our data suggest that interleukin-1ß may be instrumental in order to achieve the beneficial effects derived of fatty acid amide hydrolase genetic inactivation. This could be appreciated at the molecular (cytokine expression, amyloid production, plaque deposition) as well as behavioral levels (memory impairment). We here describe a previously unknown link between the endocannabinoid system and interleukin-1ß in the context of Alzheimer's disease that open new possibilities for the development of novel therapeutics.