Effects of saxitoxins exposure on oligodendrocyte development in mouse neonates.

Saxitoxins (STXs) are neurotoxins produced by cyanobacteria and dinoflagellates, and they are primarily known to block voltage-gated sodium channels in neurons. The present study aimed to obtain further information regarding the effects of these toxins on neurodevelopment by investigating the responses of murine subventricular zone (SVZ) neural progenitors to STXs. An in vitro neonatal mouse SVZ explant model was exposed to different concentrations of toxic cyanobacterial extracts to evaluate the migration and differentiation of SVZ-derived progenitor cells. To test the ability of STX to cross the placental barrier, pregnant mice received a single intraperitoneal injection of STXs (7.5 µg/kg body weight) on gestational day fifteen. Immunocytochemistry was performed to detect proliferating and differentiating progenitors, including oligodendrocyte progenitor cells (OPCs). It was found that specific proliferation of OPCs was significantly increased, but there was no corresponding increase in the number of differentiated oligodendrocytes, which may indicate a negative effect on the maturation process of these cells. Additionally, the data showed that STXs crossed the placental barrier. Thus, STXs can be considered a potential risk to fetal neurodevelopment.

Astrocyte heterogeneity in the brain: from development to disease.

In the last decades, astrocytes have risen from passive supporters of neuronal activity to central players in brain function and cognition. Likewise, the heterogeneity of astrocytes starts to become recognized in contrast to the homogeneous population previously predicted. In this review, we focused on astrocyte heterogeneity in terms of their morphological, protein expression and functional aspects, and debate in a historical perspective the diversity encountered in glial progenitors and how they may reflect mature astrocyte heterogeneity. We discussed data that show that different progenitors may have unsuspected roles in developmental processes. We have approached the functions of astrocyte subpopulations on the onset of psychiatric and neurological diseases.

Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: cross talk between glycero- and sphingolipids in the kidney.

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the µM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Lysophosphatidic acid can support the formation of membranous structures and an increase in MBP mRNA levels in differentiating oligodendrocytes.

During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both the receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes' process network, but only when PD-Ialpha/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation.

Phosphodiesterase-Ialpha/autotaxin's MORFO domain regulates oligodendroglial process network formation and focal adhesion organization.

Development of a complex process network by maturing oligodendrocytes is a critical but currently poorly characterized step toward myelination. Here, we demonstrate that the matricellular oligodendrocyte-derived protein phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX) and especially its MORFO domain are able to promote this developmental step. In particular, the single EF hand-like motif located within PD-Ialpha/ATX's MORFO domain was found to stimulate the outgrowth of higher order branches but not process elongation. This motif was also observed to be critical for the stimulatory effect of PD-Ialpha/ATX's MORFO domain on the reorganization of focal adhesions located at the leading edge of oligodendroglial protrusions. Collectively, our data suggest that PD-Ialpha/ATX promotes oligodendroglial process network formation and expansion via the cooperative action of multiple functional sites located within the MORFO domain and more specifically, a novel signaling pathway mediated by the single EF hand-like motif and regulating the correlated events of process outgrowth and focal adhesion organization.

Phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX): a multifunctional protein involved in central nervous system development and disease.

Phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX) was originally identified as a cell-motility-stimulating factor secreted by a variety of tumor cells. Thus, studies related to its potential functional roles have traditionally focused on tumorigenesis. PD-Ialpha/ATX's catalytic activity, initially defined as nucleotide pyrophosphatase/phosphodiesterase, was soon recognized as being necessary for its tumor cell-motility-stimulating activity. However, only the discovery of PD-Ialpha/ATX's identity with lysophospholipase D, an extracellular enzyme that converts lysophosphatidylcholine into lysophosphatidic acid (LPA) and potentially sphingosylphosphoryl choline into sphingosine 1-phosphate (S1P), revealed the actual effectors responsible for PD-Ialpha/ATX's ascribed motogenic functions, i.e., its catalytic products. PD-Ialpha/ATX has also been detected during normal development in a number of tissues, in particular, the central nervous system (CNS), where expression levels are high. Similar to tumor cells, PD-Ialpha/ATX-expressing CNS cells secrete catalytically active PD-Ialpha/ATX into the extracellular environment. Thus, it appears reasonable to assume that PD-Ialpha/ATX's CNS-related functions are mediated via lysophospholipid, LPA and potentially S1P, signaling. However, recent studies identified PD-Ialpha/ATX as a matricellular protein involved in the modulation of oligodendrocyte-extracellular matrix interactions and oligodendrocyte remodeling. This property of PD-Ialpha/ATX was found to be independent of its catalytic activity and to be mediated by a novel functionally active domain. These findings, therefore, uncover PD-Ialpha/ATX, at least in the CNS, as a multifunctional protein able to induce complex signaling cascades via distinct structure-function domains. This Mini-Review describes PD-Ialpha/ATX's multifunctional roles in the CNS and discusses their potential contributions to CNS development and pathology.

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C.

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.

Sphingosine-1-phosphate formation activates phosphatidylinositol-4 kinase in basolateral membranes from kidney cells: crosstalk in cell signaling through sphingolipids and phospholipids.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.