SUMMARY OF THE RESEARCH PROJECT

The endocannabinoid system (ECS) is composed of several elements, including cell membrane receptors (CB1, TRPV1, GPR55), exogenous ligands (such as delta-9-tetrahydrocannabinol, cannabidiol, etc.) and endogenous (known as “endocannabinnoids” among which anandamide, AEA and 2-arachidonoylglycerol, 2-AG are most relevant), as well as the enzymatic machinery of synthesis and degradation of these ligands (i.e. fatty acid amide hydrolase, FAAH, which degrades AEA and the monoglyceride lipase, MGL, which in turn degrades 2-AG).

Neuroinflammation is a severe condition that is key in multiple neurodegenerative processes, including Alzheimer´s disease (AD). The neuroinflammatory reaction of this disease is caused by the formation of β-amyloid peptide oligomers 1-42 and 1-40 and their clustering in neuritic plaques in the brain parenchyma. These pathologic structures, together with the formation of neurofibrillary tangles -aggregates of hyperphosphorylated tau protein – constitute the main characteristics of AD. Currently, the ECS is generally considered part of the endogenous mechanisms of neuroprotection within the CNS and is regarded as a possible target to treat neuroinflammation.

Our research group is focused on two main lines of investigation. On the one hand, the effect derived from the inactivation of FAAH enzyme in the context of AD; and on the other hand, the role of the cannabinoid CB2 receptor in neuroinflammatory processes. Recent findings in our laboratory highlight the relevance of an increase in the levels of AEA as regards to: (i) severe brain damage; and (ii) chronic neuroinflammation. We have observed that an increase in the levels of AEA through the genetic inactivation of its degradative enzyme, FAAH, alters the activity of astrocytic membrane channels, increases the capacity of microglia to respond to focal injuries in the brain parenchyma and modifies the pattern of expression of inflammatory cytokines and enzymes in a murine model of amyloidosis (5xFAD). All these data indicate an increase in the inflammatory cellular environment caused by the inactivation of FAAH and the subsequent increase in the levels of AEA in the CNS, in which interleukin-1 (IL-1) seems to have a key role.

Moreover, our research group has recently developed a new experimental tool that can provide relevant insights into the role of the CB2 cannabinoid receptor in several pathological processes. This tool is a new transgenic mouse model (CB2EGFP/f/f), which has two main characteristics: i) it is a “reporter” mouse for CB2 receptor, since the expression of green fluorescent protein (GFP) is controlled by CB2 receptor´s promoter; and ii) at the same time it is a “conditional knock-out” mouse, due to the fact that the construct is flanked by two LoxP sites. After crossing this mouse line with a mouse model of Alzheimer´s disease (5xFAD), we proved our initial hypothesis, observing that the expression of CB2 receptors in a healthy brain is undetectable, while it is significantly increased in microglial cells located in the proximity of β-amyloid peptide deposits, characteristic of the animal model of the disease.
We employ a variety of experimental tools to tackle these issues, including multiphoton in vivo microscopy, molecular biology, behavioural tests, etc.

RESEARCH INTERESTS:

Several key findings from our group are indicative of a relevant role for the fatty acid amide hydrolase (FAAH), an endocannabinoid-degrading enzyme, and the cannabinoid CB2 receptor, in the context of amyloid-induced neuroinflammation, a feature of Alzheimer’s disease (AD). We have recently shown that the increase in AEA levels by blocking FAAH (main degradative enzyme for AEA and other lipids) leads to significant changes in the amyloid pathology as well as in the neuroinflammatory status in an animal model of AD (5xFAD). Our data are highly suggestive of a pathophysiologically relevant interplay among FAAH-regulated AEA levels and IL1 in the context of the neuroinflammation triggered by amyloid deposition. In light of our recently published data, we conclude that the pro-inflammatory millieu found in 5xFAD/FAAH-/- mice contibutes to the behavioral and molecular improvements observed in these mice. This improvement was evident in terms of lower memory impairment (Morris water maze, MWM) as well as in decreased levels of soluble amyloid production and deposition in the form of neuritic plaques.

Two recent observations obtained in our laboratory confirm this notion: i) the expression levels of the anti-inflammatory cytokine IL-10 was decreased in 5xFAD/FAAH-/- as compared to those in 5xFAD mice; and ii) the chronic exposure of 5xFAD/FAAH-/- mice to minocycline (10mg/kg, i.p., daily for 12 days), an inhibitor of IL1 synthesis, reverted the decrease in amyloid deposition previously observed in these mice. These results are confirmative of the beneficial effects of elevated neuroinflammation exerts in 5xFAD/FAAH-/- mice and confirms data indicating that decreased levels of IL-10 and increased levels of IL1, may delay disease progress in animal models of AD.

On the other hand, cannabinoid CB2 receptors are putative targets in AD. However, the absence of appropriate experimental tools has severely limited these studies. The new mouse models that we have recently developed (CB2eGFP/f/f and CB2-/-) allow us to tackle the study of the pathophysiological relevance of CB2 receptors from novel perspectives. By using eGFP as reporter (as its expression is under the control of the Cb2 gene promoter region), we have conducted preliminary studies on the expression pattern of CB2 receptors under physiological as well as under pathological conditions in the mouse. With these data, we have so far: i) confirmed the restricted basal expression of CB2 receptors to specific cellular elements in peripheral tissues (including spleen, lungs, liver, kidney, and thymus); ii) corroborated the negligible expression levels of CB2 receptors in the CNS (brain and spinal cord); iii) their induction (in microglial cells only) under conditions of chronic neuroinflammation in the context of AD; and iv) their potential candidacy as diagnostic markers, as this induction takes place early in the inflammatory process.

PUBLICATIONS (last 5 years)

N. Murataeva, et al. “Cannabinoid CB2 receptors are upregulated with corneal injury and regulate the course of corneal wound healing”. Experimental Eye Research 182:74-84, 2019.

A. López, et al. “Cannabinoid CB2 receptors in the mouse brain: relevance for Alzheirmer’s disease”. Journal of Neuroinflammation 15:158, 2018.

M.S. Aymerich, E. et al. “Cannabinoid Pharmacology/Therapeutics in chronic neurodegenerative diseases”. Biochemical Pharmacology 157:67-84, 2018.

J. Borowska-Fielding, et al. “Revisiting cannabinoid receptor 2 expression and function in murine retina”. Neuropharmacology 141:21-31, 2018.

N. Aparicio, et al. “Role of interleukin-1beta in the inflammatory response in a fatty acid mide hydrolase-knockout mouse model of Alzheimer’s disease”. Biochemical Pharmacology 157:202-209, 2018.

C. Vázquez, et al. “Endocannabinoids regulate the activity of astrocytic hemichannels and the microglial response against an injury: in vivo studies”. Neurobiology of Disease 79:41-50, 2015.

C. Vázquez, et al. “Endocannabinoid regulation of amyloid-induced neuroinflammation”. Neurobiology of Aging 36:3008-3019, 2015.

PROJECTS

Research project funded by the company GW-pharmaceuticals (United Kingdom) entitled: “Cannabinoid CB2 receptors and microglial pathology” (ref GWCR1636).
Research project funded by the Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad) (SAF 2016-75959-R) entitled: “Modulation of the endocannabinoid system in the context of neuroinflammation associated to Alzheimer´s disease: focus on CB2 receptor and FAAH enzyme”.

Research project funded by Francisco de Vitoria University entitled: “Phenotypic characterisation of microglia that is positive for CB2 receptor in the context of Alzheimer´s disease”

PATENTS Julian Romero Paredes. Co-inventor: “New family of 1-Indazole carboxylic derivatives with cannabinoid properties and-or cholinergic and/or regulatory of β-amyloid peptide”. WO2017/103319 A1. Spain´s Higher Council of Scientific Research (Consejo Superior de Investigaciones Cientificas – CSIC). Rey Juan Carlos University (Universidad Rey Juan Carlos) and Alcorcon Foundation University Hospital (Hospital Universitario Fundacion Alcorcon).