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In the central nervous system, oligodendrocytes synthesize the myelin, a specialized membrane to wrap axons in a discontinuous way allowing a rapid saltatory nerve impulse conduction. Oligodendrocytes express a number of growth factors and neurotransmitters receptors that allow them to sense the environment and interact with neurons and other glial cells. Depending on the cell cycle stage, oligodendrocytes may respond to these signals by regulating their survival, proliferation, migration, and differentiation. Among these signals are the endocannabinoids, lipidic molecules synthesized from phospholipids in the plasma membrane in response to cell activation. Here, we discuss the evidence showing that oligodendrocytes express a full endocannabinoid signaling machinery involved in physiological oligodendrocyte functions that can be therapeutically exploited to promote remyelination in central nervous system pathologies.

The X-linked form of Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia, is due to mutations in the ANOS1 gene that encodes for the extracellular matrix (ECM) protein anosmin 1. Prokineticins (PKs) exert their biological functions through the activation of the G protein-coupled receptors (GPCRs) prokineticin receptor 1 and 2 (PKR1, 2), and mutations in the PK2 and PKR2 genes are involved in the pathogenesis of KS. We have previously shown interaction between PKR2 and anosmin 1 in vitro. In the current report we present evidence of the modulation of PK2/PKR2 activity by anosmin 1, since this protein is able to enhance the activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) pathway elicited by PK2 through PKR2. We also show that the N-terminal region of anosmin 1, capable of binding to the PK2-binding domain of PKR2, seems to be responsible for this effect. The whey acidic protein domain (WAP) is necessary for this modulatory activity, although data from GST pull-down (glutathione-S-transferase) and analysis of the N267K mutation in the fibronectin type III domain 1 (FnIII.1) suggest the cysteine-rich (CR) and the FnIII.1 domains could assist the WAP domain both in the binding to PKR2 and in the modulation of the activation of the receptor by PK2. Our data support the idea of a modulatory role of anosmin 1 in the biological effects controlled by the PK2/PKR2 system., This research was supported by grants from the Gobierno de Castilla-La Mancha, Spain (PI2009/29) and Fundación Salud 2000, Spain (Merck-Serono Research Grant 2013) to Pedro F. Esteban.

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long-lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot-gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), voltage-dependent anion channel 1 (VDAC1), myosin phosphatase Rho-interacting protein (MPRIP) or usher syndrome type-1C protein-binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic-associated proteins. We describe also interacting partners specifically immunoprecipitated with either N-terminal or C-terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.

In the last years, regional differences have been reported between the brain and spinal cord oligodendrocytes, which should be considered when designing therapeutic strategies for myelin repair. Promising targets to achieve myelin restoration are the different components of the endocannabinoid system (ECS) that modulate oligodendrocyte biology, but almost all studies have been focused on brain-derived cells. Therefore, we compared the ECS between the spinal cord and cerebral cortex-derived oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (OLs). Cells from both regions express synthesizing and degrading enzymes for the endocannabinoid 2-arachidonoylglycerol, and degrading enzymes increase with maturation, more notably in the spinal cord (monoglyceride lipase-MGLL, alpha/beta hydrolase domain-containing 6-ABHD6, and alpha/beta hydrolase domain-containing 12-ABHD12). In addition, spinal cord OPCs express higher levels of the synthesizing enzymes diacylglycerol lipases alpha (DAGLA) and beta (DAGLB) than cortical ones, DAGLA reaching statistical significance. Cells from both the cortex and spinal cord express low levels of NAEs synthesizing enzymes, except for the glycerophosphodiester phosphodiesterase 1 (GDE-1) but high levels of the degrading enzyme fatty acid amidohydrolase (FAAH) that increases with maturation. Finally, cells from both regions show similar levels of CB1 receptor and GPR55, but spinal cord-derived cells show significantly higher levels of transient receptor potential cation channel V1 (TRPV1) and CB2. Overall, our results show that the majority of the ECS components could be targeted in OPCs and OLs from both the spinal cord and brain, but regional heterogeneity has to be considered for DAGLA, MGLL, ABHD6, ABHD12, GDE1, CB2, or TRPV1.

This article belongs to the Special Issue Glial Cells in Central Nervous System (CNS) Pathology and Repair., Besides giving rise to oligodendrocytes (the only myelin-forming cell in the Central Nervous System (CNS) in physiological conditions), Oligodendrocyte Precursor Cells (OPCs) are responsible for spontaneous remyelination after a demyelinating lesion. They are present along the mouse and human CNS, both during development and in adulthood, yet how OPC physiological behavior is modified throughout life is not fully understood. The activity of adult human OPCs is still particularly unexplored. Significantly, most of the molecules involved in OPC-mediated remyelination are also involved in their development, a phenomenon that may be clinically relevant. In the present article, we have compared the intrinsic properties of OPCs isolated from the cerebral cortex of neonatal, postnatal and adult mice, as well as those recovered from neurosurgical adult human cerebral cortex tissue. By analyzing intact OPCs for the first time with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, we show that these cells behave distinctly and that they have different metabolic patterns in function for their stage of maturity. Moreover, their response to Fibroblast Growth Gactor-2 (FGF-2) and anosmin-1 (two molecules that have known effects on OPC biology during development and that are overexpressed in individuals with Multiple Sclerosis (MS)) differs in relation to their developmental stage and in the function of the species. Our data reveal that the behavior of adult human and mouse OPCs differs in a very dynamic way that should be very relevant when testing drugs and for the proper design of effective pharmacological and/or cell therapies for MS., This research was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades (former Ministerio de Economía y Competitividad-MINECO: grant numbers SAF2009–07842, ADE10-0010, SAF2012-40023, SAF2016-77575-R, and the Red Española de Esclerosis Múltiple: RD12-0032-12; RD16/0015/0019), Spanish Research Council-CSIC (grant number PID2019-109858RB-100), the ‘‘Fundación Eugenio Rodríguez Pascual’’ (Spain) and the “Fundación Ramón Areces” (grant number CIVP19A5917). The APC was funded by grants SAF2016-77575-R and PID2019-109858RB-100.

While the endocannabinoid 2-arachidonoylglycerol (2-AG) is thought to enhance the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) in vitro, less is known about how endogenous 2-AG may influence the migration of these cells. When we assessed this in Agarose drop and Boyden chemotaxis chamber assays, inhibiting the sn-1-diacylglycerol lipases α and β (DAGLs) that are responsible for 2-AG synthesis significantly reduced the migration of OPCs stimulated by platelet-derived growth factor-AA (PDGF) and basic fibroblast growth factor (FGF). Likewise, antagonists of the CB1 and CB2 cannabinoid receptors (AM281 and AM630, respectively) produced a similar inhibition of OPC migration. By contrast, increasing the levels of endogenous 2-AG by blocking its degradation (impairing monoacylglycerol lipase activity with JZL-184) significantly increased OPC migration, as did agonists of the CB1, CB2 or CB1/CB2 cannabinoid receptors. This latter effect was abolished by selective CB1 or CB2 antagonists, strongly suggesting that cannabinoid receptor activation specifically potentiates OPC chemotaxis and chemokinesis in response to PDGF/FGF. Furthermore, the chemoattractive activity of these cannabinoid receptor agonists on OPCs was even evident in the absence of PDGF/FGF. In cultured brain slices prepared from the corpus callosum of postnatal rat brains, DAGL or cannabinoid receptor inhibition substantially diminished the in situ migration of Sox10+ OPCs. Overall, these results reveal a novel function of endogenous 2-AG in PDGF and FGF induced OPC migration, highlighting the importance of the endocannabinoid system in regulating essential steps in oligodendrocyte development.

Cannabinoid receptor 1 (CBWe use CBSample heating above 65 °C greatly impaired CBWe obtain consistent and specific CBWe establish experimental guidelines for the detection of CB

Sexual maturation and olfactory bulb defects found in prokineticin 2 (Pk2) and prokineticin receptor 2 (Pkr2) mutant mice resembling the phenotypic characteristics of Kallmann syndrome (KS), gave rise to the question of whether these genes would have a role in KS pathogenesis. Later, mutations in both genes were identified in patients suffering from KS. The gene responsible for the Xlinked form of KS, ANOS1, encodes the ECM protein anosmin 1. Among other functions, anosmin 1 can regulate the activity of FGFR1, encoded by one of the genes involved in the autosomal transmission of KS. Therefore, it has been proposed that anosmin 1 could interact with PKR2 to modulate its activity. We present the first evidence supporting this hypothesis and report the interaction of full-length anosmin 1 with three extracellular domains of PKR2. A truncated anosmin 1 protein comprising the first three domains of the protein interacts with the second extracellular loop of PKR2, involved in PK2 binding. Finally, last three FnIII repeats of anosmin 1 also interacted with the PKR2 domains that interacted with full-length anosmin 1. Our data represent a molecular link between two of the genes involved in KS pathogenesis., This research was supported with grants from the Gobierno de Castilla-La Mancha, Spain (PI2009/29) and Fundación Salud 2000, Spain (Merck-Serono Research Grant 2013) to PFE. VMB was a PhD student hired by Gobierno de Castilla-La Mancha (MOV2007-JI/19). PFE was a researcher hired by SESCAM, Gobierno de Castilla-La Mancha, Spain and with funds from the Spanish Ministerio de Economía, Innovación y Competitividad MINECO (ADE10-0010) granted to Fernando de Castro, Neurobiología del Desarrollo group, Hospital Nacional de Parapléjicos (Toledo, Spain).

Background Cannabinoid receptor 1 (CB1) identification by western blot (WB) has generated a great deal of controversial data making the interpretation of the results difficult. Our purpose is to find the most adequate experimental conditions to detect CB1 by WB and immunoprecipitation (IP) as a first step towards the study of CB1 interactome. New method We use CB1 knockout mice tissue as negative controls and describe appropriate sample handling conditions for CB1 detection by WB and IP from brain and cortical neuron cultures. Results Sample heating above 65 °C greatly impaired CB1 detection by WB, since it favored the formation of high molecular weight aggregates. We also show the convenience of using n-dodecyl-β- d -maltoside (DDM) as a detergent for the detection of CB1 by WB and, mostly, for IP. Comparison with existing method(s) We obtain consistent and specific CB1 detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB1 identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods. Conclusions We establish experimental guidelines for the detection of CB1 by WB and the study of CB1 interacting proteins by IP. We propose a new interpretation of CB1 WB and IP data based on the folding and packing state of the protein and the detergent used. The standardization of the most advantageous conditions for coimmunoprecipitation (CoIP) would be a useful tool for the future study of the interactome of CB1.

New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB., This research was supported by grants from the Spanish Ministerio de Economía, Innovación y Competitividad MINECO (SAF2009-07842, ADE10-0010, RD07-0060-2007, RD12-0032-12 and SAF2012-40023 to FdC; and BFU2010-18284 to JMG-V), FISCAM (Gobierno de Castilla-La Mancha, Spain—Grant Number PI2007-66), the Junta de Castilla y León (Spain, to EW), and from the Fundación Eugenio Rodríguez Pascual (Spain) to FdC. DGG and VMB were PhD students hired by Gobierno de Castilla-La Mancha (MOV2010-JI/11 and MOV2007-JI/19, respectively). FdCS is a CSIC staff scientist in special permission hired by SESCAM (Gobierno de Castilla-La Mancha, Spain). PFE was a researcher hired by SESCAM (Gobierno de Castilla-La Mancha) and ADE10-0010.

During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad-MINECO (Grant Numbers SAF2009-07842, SAF2012-40023, RD07-0060-2007 and RD12-0032-12 Red Española de Esclerosis Múltple) and the “Fundación Eugenio Rodríguez Pascual” to FdC; the Fundación para la Investigación Socio-sanitaria de Castilla La-Mancha FISCAM (Grant Number PI2007-66 to FdC and PI2009-29 to PFE); from the Spanish MINECO (Grant Numbers BFU2008-00899 and BFU2008-03390) and the Junta de Andalucía (Grant Numbers BIO-122, CVI-02487 and P07-CVI-02686) to JMDG and AGM; and from the Spanish MINECO (Grant Number BFU-2009-08404) to RL. VMB was hired under FISCAM (MOV2007-JI/19), SAF2009-07842 and RD07-0060-2007; PFE was hired by the Servicio de Salud de Castilla La-Mancha (SESCAM, Gobierno de Castilla-La Mancha) and under grant ADE10-0010 from MINECO to FdC. FdC is a CSIC Staff Scientist on leave of absence and currently hired by SESCAM.

During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination.