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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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
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•Anosmin 1 interacts with PKR2, suggesting it could modulate the receptor activity.•Mutations found in Kallmann syndrome patients disrupt the interaction.•Anosmin 1 N-terminal domains interact with the ligand-binding domain of PKR2.•Anosmin 1 N-terminal domains modulate PKR2 activation by PK2.•Anosmin 1 could regulate the PK2/PKR2 system in normal and pathological conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Main Points
In oligodendroglial cells there is an operational endocannabinoid signaling network.
2‐AG contributes to the proliferation, directional migration and maturation of OPCs.
Manipulation of the endocannabinoid system exert beneficial therapeutic effects.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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•We provide precise experimental guidelines for CB1 detection by Western blot and immunoprecipitation.•We describe the formation of high molecular weight CB1 aggregates after ...heating.•We identify n-dodecyl-β-d-maltoside, DDM, as a preferred detergent for CB1 detection by WB and IP.•Based on WB, IP and deglycosylation results, we propose a new interpretation of CB1 WB and IP data.
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.
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.
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.
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.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Cannabinoids are lipid mediators that participate in brain development. Appropriate activation and signaling of CB1 cannabinoid receptor 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 describe new molecular partners that associate with CB1 during neuronal postnatal maturation and may mediate the different cannabinoid actions along with the development of brain circuits.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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.
The risk of complications following surgical procedures is significantly increased in patients with SARS-CoV-2 infection. However, the mechanisms underlying these correlations are not fully known. ...Spinal cord injury (SCI) patients who underwent reconstructive surgery for pressure ulcers (PUs) before and during the COVID-19 pandemic were included in this study. The patient's postoperative progression was registered, and the subcutaneous white adipose tissue (s-WAT) surrounding the ulcers was analyzed by proteomic and immunohistochemical assays to identify the molecular/cellular signatures of impaired recovery. Patients with SCI and a COVID-19-positive diagnosis showed worse recovery and severe postoperative complications, requiring reintervention. Several proteins were upregulated in the adipose tissue of these patients. Among them, CKMT2 and CKM stood out, and CKM increased for up to 60 days after the COVID-19 diagnosis. Moreover, CKMT2 and CKM were largely found in MGCs within the s-WAT of COVID patients. Some of these proteins presented post-translational modifications and were targeted by autoantibodies in the serum of COVID patients. Overall, our results indicate that CKMT2, CKM, and the presence of MGCs in the adipose tissue surrounding PUs in post-COVID patients could be predictive biomarkers of postsurgical complications. These results suggest that the inflammatory response in adipose tissue may underlie the defective repair seen after surgery.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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