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ENDOCANNABINOID AND NEUROINFLAMMATION GROUP

STAFF

PRINCIPAL INVESTIGATOR:

Dr. Julián Romero Paredes j.romero.prof@ufv.es

RESEARCHERS:

María Teresa Grande Rodríguez
Ana María Martínez Relimpio
Rocío Palenzuela Muñoz
María Posada Ayala
Samuel Ruiz de Martín Esteban

PREDOCTORAL STUDENTS:

Andrea Arnanz Paredes
Laura Martín Pérez
Almudena López Escobar

RESEARCH SUMMARY

The endocannabinoid system (ECS) is made up of various elements, including cell membrane receptors (CB1, CB2, TRPV1, GPR55), endogenous ligands (called “endocannabinoids”, among which anandamide, AEA, and 2-arachidonylglycerol, etc.) and exogenous ligands (such as delta-9-tetrahydrocannabinol, cannabidiol, etc.), as well as the enzymatic machinery of synthesis and degradation of these ligands, 2-AG) and exogenous (such as delta-9-tetrahydrocannabinol, cannabidiol, etc), as well as the enzymatic machinery of synthesis and degradation of these ligands (such as fatty acid amide hydrolase, FAAH, which degrades AEA, and monoglyceride lipase, MGL, which degrades 2-AG).

Neuroinflammation is a key condition in many neurodegenerative processes, including Alzheimer’s disease (AD). The neuroinflammatory reaction in this disease is caused by the formation of amyloid beta peptide (Aβ) 1-42 and 1-40 oligomers and their deposition in the form of neuritic plaques in the brain parenchyma. These pathological structures, together with the formation of neurofibrillary tangles of hyperphosphorylated tau protein, constitute the main features of AD. Nowadays, SEC is generally considered to be appreciated as part of the endogenous neuroprotective mechanisms available to the CNS and is postulated as a possible target for the treatment of neuroinflammation.

Our research group focuses on two main lines: on the one hand, on the effect derived from the inactivation of the FAAH enzyme in the context of AD and, on the other hand, on the role of the cannabinoid CB2 receptor in neuroinflammatory processes. First, recent data obtained in our laboratory underline the relevance of an increased tone of AEA in relation to: i) basal glial cell activity; ii) acute brain damage; and iii) chronic neuroinflammation. Thus, we have observed that an increase in AEA levels through genetic inactivation of its degradative enzyme, FAAH, alters the activity of astrocyte membrane hemichannels, increases microglial responsiveness after focal brain parenchymal damage and modifies the expression pattern of inflammatory cytokines and enzymes in a murine model of amyloidosis (5xFAD). All these data point to an elevation in the inflammatory cellular environment derived from FAAH inactivation and subsequent increase in AEA levels in the CNS in which interleukin-1 (IL-1) seems to play a prominent role.

On the other hand, our group has recently developed a new experimental tool that can provide relevant data about the role of the cannabinoid CB2 receptor in several pathological processes. This tool is a new transgenic mouse model (CB2eGFP/f/f) that has two fundamental characteristics: i) it is a “reporter” mouse for the CB2 receptor, since the expression of the green fluorescent protein (GFP) takes place under the control of the CB2 receptor promoter region; and, ii) it is, at the same time, a “knockout-conditional” mouse, since the construct used is flanked by two loxp sites. Once we crossed this mouse line with another model of Alzheimer’s disease (5xFAD), we were able to corroborate our hypothesis by observing that CB2 receptor expression in the healthy brain is undetectable while it is significantly increased in the microglia cells located in the vicinity of the beta-amyloid peptide deposits characteristic of this animal model of the disease.

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.

PROJECTS:

Research project funded by GW-Pharmaceuticals (UK) entitled: “Cannabinoid CB2 receptors and microglial pathophysiology” (ref GWCR1636).

Research project funded by the Spanish Ministry of Economy and Competitiveness (SAF 2016-75959-R) with title: “Modulation of the endocannabinoid system in the context of Alzheimer’s disease-associated neuroinflammation: focus on the CB2 receptor and the FAAH enzyme”.

Research project funded by the Universidad Francisco de Vitoria entitled: “Phenotypic characterization of CB2 cannabinoid receptor-positive microglia in the context of Alzheimer’s disease”.

Research project funded by the Universidad Francisco de Vitoria entitled: “Neuroinflammation in the context of Alzheimer’s disease: role of the FAAH enzyme in microglial activity”.

Research project funded by the Ministry of Science and Innovation (SAF PID2019-108992RB-I00) entitled: “The cannabinoid CB2 receptor and the FAAH enzyme as therapeutic targets in Alzheimer’s disease: glia, neuroinflammation and new experimental tools”.

Research project funded by the Alicia Koplowitz Foundation entitled: “Towards a “biosignature” of self-injurious behaviors (suicidal and non-suicidal) in childhood and adolescence: role of the endocannabinoid system and beta-endorphin in peripheral blood”.

PATENTS
Julián Romero Paredes. Co-inventor: “New family of 1-Indazolyl carbonyl derivatives with cannabinoid and/or cholinergic and/or beta-amyloid peptide regulatory properties”. WO2017/103319 A1. Consejo Superior de Investigaciones Científicas, Universidad Rey Juan Carlos and Hospital Universitario Fundación Alcorcón.

Last five years PUBLICATIONS

Xiaoyan Li, Zhili Xu, Lawrence Carey, Julián Romero, Alexandros Makryiannis, Cecilia J. Hillard, Philip Albrecht, Feank Rice, Ken Mackie, Andrea G. Hohmann. “Peripheral cannabinoid CB2 receptors mechanisms suppress chemotherapy-induced peripheral neuropathy: Evidence from a CB2 reporter mouse”. Pain2021 Sep 24. doi: 10.1097/j.pain.0000000000002502. Online ahead of print. PMID: 35001054

Thais Gazzi, Benjamin Brennecke, Kenneth Atz, Claudia Korn, David Sykes, Gabriel Forn-Cuni, Patrick Pfaff, Roman C. Sarott, Matthias V. Westphal, Yelena Mostinski, Leonard Mach, Malgorzata Wasinska-Kalwa, Marie Weise, Bradley L. Hoare, Tamara Miljuš, Maira Mexi, Nicolas Roth, Eline J. Koers, Wolfgang Guba, André Alker, Arne C. Rufer, Eric A. Kusznir, Sylwia Huber, Catarina Raposo, Elisabeth A. Zirwes, Anja Osterwald, Anto Pavlovic, Svenja Moes, Jennifer Beck, Matthias Nettekoven, Irene Benito-Cuesta, Teresa Grande, Faye Drawnel, Gabriella Widmer, Daniela Holzer, Tom van der Wel, Harpreet Mandhair, Yurii Saroz, Natasha Grimsey, Michael Honer, Jürgen Fingerle, Jörg Scheffel, Johannes Broichhagen, Klaus Gawrisch, Julián Romero, Cecilia J. Hillard, Zoltan V. Varga, Mario van der Stelt, Pal Pacher, Jürg Gertsch, Christoph Ullmer, Peter J. McCormick, Sergio Oddi, Herman P. Spaink, Mauro Maccarrone, Dmitry B. Veprintsev, Erick M. Carreira, Uwe Grether, Marc Nazaré. “Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe”. Chemical Science (in press).

A. López, N. Aparicio, M.R. Pazos, M.T. Grande, M.A. Barreda-Manso, I. Benito-Cuesta, C. Vázquez, M. Amores, G. Ruiz-Pérez, E. García-García, M. Beatka, R.M. Tolón, B.N. Dittel, C.J. Hillard, J. Romero. “Cannabinoid CB2 receptors in the mouse brain: relevance for Alzheirmer’s disease”. Journal of Neuroinflammation 15:158, 2018.

M.S. Aymerich, E. Aso, M.A. Abellanas, R.M. Tolon, J.A. Ramos, I. Ferrer, J. Romero, J Fernandez-Ruiz. “Cannabinoid Pharmacology/Therapeutics in chronic neurodegenerative diseases”. Biochemical Pharmacology 157:67-84, 2018.

J. Borowska-Fielding, N. Murataeva, B. Smith, A.M. Szczesniak, E. Leishmann, L. Daily, J.T. Toguri, C.J. Hillard, J. Romero, H.B. Bradshaw, M. Kelly, A.J. Straiker. “Revisiting cannabinoid receptor 2 expression and function in murine retina”. Neuropharmacology 141:21-31, 2018.

N. Aparicio, M.T. Grande, S. Ruiz de Martín Esteban, A. López, G. Ruiz-Pérez, M. Amores, C. Vázquez, A.M. Martínez Relimpio, M.R. Pazos, B.F. Cravatt, R.M. Tolón, J. Romero. “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.

Natalia Murataeva, Sally Miller, Amey Dhopeshwarkar, Emma Leishman, Laura Daily, Xavier Taylor, Brian Morton, Matthew Lashmet, Heather Bradshaw, Cecilia Hillard, Julián Romero and Alex Straiker. “Cannabinoid CB2 receptors are upregulated with corneal injury and regulate the course of corneal wound healing”. Experimental Eye Research 182:74-84, 2019.

Roman C. Sarott, Matthias V. Westphal, Patrick Pfaff, Claudia Korn, David Sykes,Thais Gazzi, Benjamin Brennecke, Kenneth Atz, Marie Weise, Yelena Mostinski, Pattarin Hompluem, Eline Koers, Tamra Miljus, Nicolas J. Roth, Hermon Asmelash, Man C. Vong, Jacopo Piovesan, Wolfgang Guba, Arne C. Rufer, Eric A. Kusznir, Sylwia Huber, Catarina Raposo, Elisabeth A. Zirwes, Anja Osterwald, Anto Pavlovic, Svenja Moes, Jennifer Beck, Irene Benito-Cuesta, Teresa Grande, Samuel Ruiz de Martin Esteban, Alexei Yeliseev, Faye Drawnel, Gabriella Widmer, Daniela Holzer, Tom van der Wel, Harpreet Mandhair, Cheng-Yin Yuan, William R. Drobyski, Yurii Saroz,Natasha Grimsey, Michael Honer, Jürgen Fingerle, Klaus Gawrisch, Julián Romero, Cecilia J. Hillard, Zoltan V. Varga, Mario van der Stelt, Pal Pacher, Jüerg Gertsch, Peter J. McCormick, Christoph Ullmer, Sergio Oddi, Mauro Maccarrone, Dmitry B. Veprintsev, Marc Nazaré, Uwe Grether, and Erick M. Carreira. “Development of high-specificity fluorescent probes to enable cannabinoid type 2 receptor studies in living cells”. Journal of the American Chemical Society 142:16953-16964, 2020.

Cheng Yin-Yuan, Vivian Zhou, Garrett Sauber, Todd Stollenwerk, Richard Komorowski, Alicia López, Rosa M. Tolón, Julián Romero, Cecilia J. Hillard, William Drobyski. “Signaling through the type 2 cannabinoid receptor regulates the severity of acute and chronic graft versus host disease”. Blood 137:1241-1255, 2021.

Carmen Rodríguez-Cueto, Marta Gómez-Almería, Laura García Toscano, Julián Romero, Cecilia J. Hillard, Eva de Lago, Javier Fernández-Ruiz. “Inactivation of the CB2 receptor accelerated the neuropathological deterioration in TDP-43 transgenic mice, a model of amyotrophic lateral sclerosis”. Brain Pathology 13:e12972, 2021.

Gonzalo Ruiz, Samuel Ruiz de Martín Esteban, Sharai Marqués, Noelia Aparicio, M. Teresa Grande, Irene Benito-Cuesta, Ana M. Martínez Relimpio, M. Andrea Arnanz, Rosa M. Tolón, María Posada-Ayala, Benjamin F. Cravatt, José Antonio Esteban, Julián Romero, Rocío Palenzuela. “Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer’s disease”. Journal of Neuroinflammation 18:223, 2021.

Samuel Ruiz de Martín Esteban, Irene Benito-Cuesta, Itziar Terradillos, Ana M. Martínez-Relimpio, M. Andrea Arnanz, Gonzalo Ruiz-Pérez, Claudia Korn, Catarina Raposo, Roman C. Sarott, Mathias V. Westphal, Izaskun Elezgarai, Erick M. Carreira, Cecilia J. Hillard, Uwe Grether, Pedro Grandes, M. Teresa Grande, Julián Romero. “Cannabinoid CB2 receptors modulate microglia function and amyloid dynamics in a mouse model of Alzheimer’s disease”. Frontiers in Pharmacology 13:841766, 2022.

Marc Ten-Blanco, África Flores, Inmaculada Pereda-Pérez, Fabiana Piscitelli, Cristina Izquierdo-Luengo, Luigia Cristino, Julián Romero, Cecilia J. Hillard, Rafael Maldonado, Vincenzo Di Marzo, Fernando Berrendero. “Amygdalar CB2 cannabinoid receptors mediate fear extinction deficits promoted by orexin-A/hypocretin-1”. Biomedicine and Pharmacotherapy 149:112925, 2022.

Alicia Sánchez-Sanz, María Posada-Ayala, Julia Sabín-Muñoz, Ismael Fernández-Miranda, Yolanda Aladro-Benito, Roberto Álvarez-Lafuente, Ana Royuela, Ruth García-Hernández, Ofir Rodríguez-De la Fuente, Julián Romero, Antonio García-Merino, Antonio José Sánchez-López. “Endocannabinoid levels in peripheral blood mononuclear cells of multiple sclerosis patients treated with dimethyl fumarate. A longitudinal study”. Scientific Reports (in press).

Itziar Terradillos, Itziar Bonilla-Del Río, Maitane Serrano, Amaia Mimenza, Leire Lekunberri, Ilazki Anaut-Lusar, Nagore Puente, Inmaculada Gerrikagoitia, Samuel Ruiz de Martín Esteban, Cecilia J Hillard, M. Teresa Grande, Julián Romero, Izaskun Elezgarai, Pedro Grandes. “Altered glial expression of the cannabinoid CB1 receptor in the subiculum of a mouse model of Alzheimer’s disease”. Glia (in press).

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