The aim of this review is to present a modern view of the endocannabinoid system, in order to discuss in a better perspective available data from preclinical studies on the use of eCBs as new neuroprotective agents, potentially useful to prevent glaucoma and retinal neurodegenerative diseases.

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5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe 3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe 3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe 3+ , as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

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Chronic inflammation in Alzheimer’s disease (AD) has been recently identified as a major contributor to disease pathogenesis. Once activated, microglial cells, which are brain-resident immune cells, exert several key actions, including phagocytosis, chemotaxis, and the release of pro- or anti-inflammatory mediators, which could have opposite effects on brain homeostasis, depending on the stage of disease and the particular phenotype of microglial cells. The endocannabinoids (eCBs) are pleiotropic bioactive lipids increasingly recognized for their essential roles in regulating microglial activity both under normal and AD-driven pathological conditions. Here, we review the current literature regarding the involvement of this signalling system in modulating microglial phenotypes and activity in the context of homeostasis and AD-related neurodegeneration.

Growing evidence shows that the immune system is critically involved in Alzheimer’s disease (AD) pathogenesis and progression. The modulation and targeting of peripheral immune mechanisms are thus promising therapeutic or preventive strategies for AD. Given the critical involvement of the endocannabinoid (eCB) system in modulating immune functions, we investigated the potential role of the main elements of such a system, namely type-1 and type-2 cannabinoid receptors (CB1 and CB2), and fatty acid amide hydrolase (FAAH), in distinct immune cell populations of the peripheral blood of AD patients. We found that, compared to healthy controls, CB1 and CB2 expression was significantly lower in the B-lymphocytes of AD patients. Moreover, we found that CB2 was significantly lower and FAAH was significantly higher in monocytes of the same subjects. In contrast, T-lymphocytes and NK cells did not show any variation in any of these proteins. Of note, monocytic CB2 and FAAH levels significantly correlated with clinical scores. Furthermore, the pharmacological inactivation of FAAH in monocytes and monocyte-derived macrophages obtained from AD patients was able to modulate their immune responses, by reducing production of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12, and enhancing that of the anti-inflammatory cytokine IL-10. Furthermore, FAAH blockade skewed AD monocyte-derived macrophages towards a more anti-inflammatory and pro-resolving phenotype. Collectively, our findings highlight a central role of FAAH in regulating AD monocytes/macrophages that could be of value in developing novel monocyte-centered therapeutic approaches aimed at promoting a neuroprotective environment.

In this study, we investigated the role of CB palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB receptor significantly reduced its association with Gα . The agonist stimulation induced a partial dissociation of Gα proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gα from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gα and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB receptor may offer a coordinated process of regulation and could open new therapeutic approaches.

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