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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.

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.

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.

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.

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration, as well as differentiation events such as myelination. Anosmin-1 is an extracellular matrix (ECM) glycoprotein that interacts with the fibroblast growth factor receptor 1 (FGFR1) to exert its biological actions through this receptor, although the intracellular pathways underlying anosmin-1 signaling remain largely unknown. This protein is defective in the X-linked form of Kallmann syndrome (KS) and has a prominent role in the migration of neuronal and oligodendroglial precursors. We have shown that anosmin-1 exerts a chemotactic effect via FGFR1 on neuronal precursors from the subventricular zone (SVZ) and the essential role of the ERK1/2 signaling. We report here the positive chemotactic effect of FGF2 and anosmin-1 on rat and mouse postnatal OPCs via FGFR1. The same effect was observed with the truncated N-terminal region of anosmin-1 (A1Nt). The introduction in anosmin-1 of the missense mutation F517L found in patients suffering from KS annulled the chemotactic activity; however, the mutant form carrying the disease-causing mutation E514K also found in KS patients, behaved as the wild-type protein. The chemoattraction exhibited by FGF2 and anosmin-1 on OPCs was blocked by the mitogen-activated protein kinase (MAPK) inhibitor U0126, suggesting that the activation of the ERK1/2 MAPK signaling pathway following interaction with the FGFR1 is necessary for FGF2 and anosmin-1 to exert their chemotactic effect. In fact, both proteins were able to induce the phosphorylation of the ERK1/2 kinases after the activation of the FGFR1 receptor.

J. Neurochem. (2010) 115, 1256–1265. Abstract Anosmin-1, defective in Kallmann’s syndrome, participates in the adhesion, migration and differentiation of different cell types in the CNS. Although not fully understood, the mechanisms of action of Anosmin-1 involve the interaction with different proteins, being the interaction with fibroblast growth factor receptor 1 (FGFR1) and the modulation of its signalling the best studied to date. Using glutathione-S-transferase pull-down assays we demonstrate that the FnIII.3 (Fibronectin-like type III) domain and the combination whey acidic protein-FnIII.1, but not each of them individually, interact with FGFR1. The interaction of the whey acidic protein-FnIII.1 domains is substantially reduced when the cysteine-rich region is present, suggesting a likely regulatory role for this domain. The introduction in FnIII.3 of any of the two missense mutations found in Kallmann’s syndrome patients, E514K and F517L, abolished the interaction with FGFR1, what suggests an important role for these residues in the interaction. Interestingly, the chemoattraction of Anosmin-1 on rat neuronal precursors (NPs) via FGFR1 is retained by the N-terminal region of Anosmin-1 but not by FnIII.3 alone, and is lost in proteins carrying either one of the missense mutations, probably because of a highly reduced binding capacity to FGFR1. We also describe homophilic interaction Anosmin-1/Anosmin-1 via the FnIII repeats 1 and 4, and the interaction of FnIII.1 and FnIII.3 with Fibronectin and of FnIII.3 with Laminin.

International audience; At embryonic stages of development, oligodendrocyte precursors (OPCs) generated in the preoptic area colonize the entire optic nerve (ON). Different factors controlling migration of ON OPCs have been identified, including secreted growth factors, morphogens and guidance cues, as well as cell adhesion molecules. We have shown previously that the soluble form of the extracellular matrix (ECM) protein anosmin-1, impairs OPC migration induced by FGF-2. In the present work, we show that anosmin-1 is expressed by both migrating OPCs and axons of the retinal ganglion cells in the embryonic ON. In vitro, we observe that OPC migration is strongly impaired by contact with anosmin-1 when used as a substrate and, in contrast to previous results, this effect is independent of FGF-2/FGFR1 signaling. We also show that OPCs preferentially adhere to anosmin-1 when compared with other ECM molecules used as substrates, and that when the endogenous anosmin-1 expressed by OPCs is blocked, OPC adhesion to all the different substrates (including anosmin-1), is significantly reduced. This novel effect of anosmin-1 on cell adhesion is also independent of FGF-2/FGFR1. We finally demonstrate that the blockade of the endogenous anosmin-1 expressed by OPCs impairs their migration. Our data suggest that the endogenous anosmin-1 expressed by OPCs is necessary for the correct adhesion of these cells to the different components of the ECM (including anosmin-1 itself), contributing to the migration of these cells.

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.

Anosmin-1 is the glycoprotein encoded by the KAL1 gene and part of the extracellular matrix, which was first identified as defective in human Kallmann syndrome (KS, characterised by hypogonadotropic hypogonadism and anosmia); biochemically it is a cell adhesion protein. The meticulous biochemical dissection of the anosmin-1 domains has identified which domains are necessary for the protein to bind its different partners to display its biological effects. Research in the last decade has unravelled different roles of anosmin-1 during CNS development (axon pathfinding, axonal collateralisation, cell motility and migration), some of them intimately related with the cited KS but not only with this. More recently, anosmin-1 has been identified in other pathological scenarios both within (multiple sclerosis) and outside (cancer, atopic dermatitis) the CNS.

This research was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (BIO93-0161 and BIO96-1413-C02-02). P. F. Esteban has been the recipient of a fellowship from the University of Salamanca.

The molecular cloning of 1,3-beta-glucanase-encoding genes from different yeast species was achieved by screening genomic libraries with DNA probes obtained by PCR-amplification using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. The nucleotide sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 (Hansenula polymorpha) and SoEXG1 (Schwanniomyces occidentalis) genes was determined. K1EXG1 consists of a 1287 bp open reading frame encoding a protein of 429 amino acids (49,815 Da). HpEXG1 specifies a 435-amino acid polypeptide (49,268 Da) which contains two potential N-glycosylation sites. SoEXG1 encodes a protein of 425 residues (49,132 Da) which contains one potential site for N-linked glycosylation. Expression in S. cerevisiae of KlEXG1, SoEXG1 or HpEXG1 under control of their native promoters resulted in the secretion of active 1,3-beta-glucanases. Disruption of KlEXG1 did not result in a phenotype under laboratory conditions. Comparison of the primary translation products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously characterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveals that enzymes with this type of specificity constitute a family of highly conserved proteins in yeasts. KlExg1p, HpExg1p and SoExg1p contain the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases.

Anosmin-1 is the glycoprotein encoded by the KAL1 gene and part of the extracellular matrix, which was first identified as defective in human Kallmann syndrome (KS, characterised by hypogonadotropic hypogonadism and anosmia); biochemically it is a cell adhesion protein. The meticulous biochemical dissection of the anosmin-1 domains has identified which domains are necessary for the protein to bind its different partners to display its biological effects. Research in the last decade has unravelled different roles of anosmin-1 during CNS development (axon pathfinding, axonal collateralisation, cell motility and migration), some of them intimately related with the cited KS but not only with this. More recently, anosmin-1 has been identified in other pathological scenarios both within (multiple sclerosis) and outside (cancer, atopic dermatitis) the CNS.

We report the sequence of an 18,002 bp DNA fragment from the right arm of Saccharomyces cerevisiae chromosome XI. This segment contains nine complete open reading frames (ORFs), YKR401 to YKR409, and part of another ORF, YKR400, covering altogether 87·2% of the entire sequence. One of them, YKR400, encodes an NAD-dependent 5,10-methylene-tetrahydrofolate dehydrogenase. YKR404, YKR405 and YKR406 correspond to the previously characterized HBS1, MRP-L20 and PRP16 genes, coding for a translation elongation factor, a mitochondrial ribosomal protein and an ATP-binding protein, respectively. The putative product of YKR407 contains the zinc-binding region signature of neutral zinc metallopeptidases. The five other ORFs do not show significant homology to any known protein. The sequence data reported here have been assigned EMBL accession number Z27116.

As part of the EEC yeast genome program, a fragment of 15,820 bp from the right arm of Saccharomyces cerevisiae chromosome XI has been sequenced. This fragment corresponds roughly to the centromere-distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as the UBI2 gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as the MPL1 gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso1.

FGF-2 and Anosmin-1 are diffusible proteins which act in cell proliferation and/or migration during CNS development. We describe their developmental expression patterns in the subventricular zone (SVZ) of the forebrain and the neuronal precursors (NPs) that migrate from this neurogenic site towards the olfactory bulb, forming the rostral migratory stream (RMS). The analysis is carried out before (E14), during (E17, P5) and after (P15) the peaks of migration along the RMS and before this acquires its mature conformation. At all these stages, FGF-2 exerts a FGFR1-mediated motogenic effect on NPs and induces the proliferation of SVZ astrocytes (putatively type B cells from triads), and Anosmin-1 works as a typical chemotropic agent for the NPs (mediated by FGFR1 at P5-P15). Altogether, our results are consistent with the notion that FGF-2 increases cell proliferation in the SVZ and would be the motogenic cue which feeds the migration of the newly produced NPs once generated, from early development (E14) and at least until P15, while Anosmin-1 cooperates in this migration attracting the NPs. In this sense, both cues should be considered as two of the first to be chronologically identified as actors in the formation of the RMS.

11 páginas, 5 figuras, 1 tabla -- PAGS nros. 2518-2528, Anosmin-1 participates in the development of the olfactory and GnRH systems. Defects in this protein are responsible for both the anosmia and the hypogonadotrophic hypogonadism found in Kallmann's syndrome patients. Sporadically, these patients also manifest some neurological symptoms that are not explained in terms of the developmental defects in the olfactory system. We describe the pattern of Anosmin-1 expression in the central nervous system during rat development using a novel antibody raised against Anosmin-1 (Anos1). The areas with Anos1-stained neurons and glial cells were classified into three groups: (1) areas with immunoreactivity from embryonic day 16 to postnatal day (P) 15; (2) areas with Anosmin-1 expression only at postnatal development; (3) nuclei with immunoreactivity only at P15. Our data show that Anos1 immunoreactivity is detected in projecting neurons and interneurons within areas of the brain that may be affected in patients with Kallmann's syndrome that develop both the principal as well as sporadic symptoms, F.de C. was funded by the “Fondo de Investigaciones Sanitarias-FIS”/Spanish Ministerio de Sanidad, the Spanish Ministerio de Educación-MEC, partially co-financed by F.E.D.E.R., the Spanish Consejería de Sanidad/Junta de Comunidades de Castilla-La Mancha, and FISCAM. D.C. and P.F.E. were funded by grants from the Consejería de Educación y Ciencia/Junta de Comunidades de Castilla-La Mancha. F.deC. was a researcher contracted under the “Ramón y Cajal” Program (MEC), and A.B. was contracted by the “Fundación del Hospital Nacional de Parapléjicos para la Investigación y la Integración-FUNHPAIIN” (Spain)

Arf GTP-binding proteins and Rho-family GTPases play key roles in regulating membrane remodeling and cytoskeletal reorganization involved in cell movement. Several studies have implicated neurotrophins and their receptors as upstream activators of these small GTP-binding proteins, however, the mechanisms and the cell type specificity of this neurotrophin activity are still under investigation. Here we describe the rationale and protocols used for the dissection of an NT3 activated pathway that leads to the specific activation of Arf6 and Rac1.

Arf GTP-binding proteins and Rho-family GTPases play key roles in regulating membrane remodeling and cytoskeletal reorganization involved in cell movement. Several studies have implicated neurotrophins and their receptors as upstream activators of these small GTP-binding proteins, however, the mechanisms and the cell type specificity of this neurotrophin activity are still under investigation. Here we describe the rationale and protocols used for the dissection of an NT3 activated pathway that leads to the specific activation of Arf6 and Rac1.

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.

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1–tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.

Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

8 pages, 5 figures.-- et al., The Candida albicans CaENG1 gene encoding an endo-1,3-β-glucanase was cloned by screening a genomic library with a DNA probe obtained by polymerase chain reaction using synthetic oligonucleotides designed according to conserved regions found between two Saccharomyces cerevisiae endo-1,3-β-glucanases (Eng1p and Eng2p). The gene contains a 3435-bp open reading frame (ORF), capable of encoding a protein of 1145 amino acids (124,157 Da), that contains no introns. Comparison of the ScEng1p sequence with partial C. albicans genomic sequences revealed the presence of a second protein with sequence similarity (the product of the Ca20C1.22c ORF, which was named CaENG2). Disruption of the CaENG1 gene in C. albicans had no dramatic effects on the growth rate of the strains, but it resulted in the formation of chains of cells, suggesting that the protein is involved in cell separation. Expression of CaENG1 in S. cerevisiae cells afforded a 12-fold increase in the 1,3-β-glucanase activity detected in culture supernatants, showing that the protein has similar enzymatic activity to that of the S. cerevisiae Eng1p. In addition, when the C. albicans protein was expressed under its native promoter in S. cerevisiae eng1 mutant cells, it was able to complement the separation defect of this mutant, indicating that these two proteins are true functional homologues., This research was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (Grant Nos. 1FD97-1897-C02-02 and BIO2000-1573) and from the European Community (Grant No. QKL3-2000-01537). P. F. E., I. R., and R. G. were recipients of a fellowship from Ministerio de Educación y Ciencia (Spain).

International audience; The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Neurotrophin (NT)-3, one of the members of the neurotrophin family, regulates multiple events in the development and maturation of the peripheral nervous system (PNS) and the central nervous system (CNS). TrkC, the main receptor for NT-3 is expressed in the PNS, CNS, and other tissues.1 Full-length TrkC (TrkC.FL) is a approximately 150-kDa type 1 receptor tyrosine kinase protein that relays trophic signals. By alternative splicing, the trkC locus can generate truncated receptor isoforms such as TrkC.T1, which lacks the kinase domain and has a unique short intracellular domain. Overexpression of TrkC.T1 causes defects in the nervous system.2 Neurodegeneration can ensue because TrkC.T1 acts as a dominant-negative receptor of TrkC.FL or because TrkC.T1 sequesters NT-3.3,4 These mechanisms are indirect and do not require TrkC.T1 to signal. However, we recently showed that truncated Trk receptors can signal in a ligand-dependent manner, leading to the activation of Rac1 GTPase, the ruffling of the plasma membrane, and the formation of cellular protrusions.5–7 To further study the biological function of TrkC.T1 in vivo, we took advantage of the observation that the TrkC.T1 isoform is significantly upregulated during the early phase of glaucoma. TrkC.T1 upregulation was selective for glaucoma; it was not seen in optic nerve axotomy.8 We sought to determine whether TrkC.T1 was relevant to neurodegeneration in glaucoma. Glaucoma is a group of optic nerve neuropathies characterized by the chronic and progressive death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a major risk factor.9 Although the etiology of RGC death in glaucoma is multifactorial, a key contributor is the production by retinal glia of factors that are neurotoxic to RGCs. Two known neurotoxic factors are tumor necrosis factor-α (TNF-α)10–13 and α2-macroglobulin (α2m).14 These factors are secreted by the retinal glia in normal eyes and in glaucomatous eyes. However, the mechanism by which the retinal glia can finely regulate baseline secretion versus upregulated secretion of proteins that cause progressive RGC death in a chronic condition such as glaucoma is unknown.15 Therefore, we explored the mechanisms that regulate the production of neurotoxic factors that cause RGC death in glaucoma. Here we provide genetic, anatomic, and pharmacologic evidence correlating the glaucoma-induced expression of TrkC.T1 and the production of TNF-α in activated retinal glia or Muller cells leading to RGC death over time. Together, these data suggest a paracrine mechanism whereby high IOP causes early upregulation of TrkC.T1, which in turn regulates TNF-α production, causing glaucomatous RGC death. This work provides new evidence on the relevance of truncated neurotrophin receptors in disease and potentially validates TrkC.T1 as a target for glaucoma therapy.