Brain-derived neurotrophic factor administration mediated oligodendrocyte differentiation and myelin formation in subcortical ischemic stroke.

BACKGROUND AND PURPOSE: Translational research is beginning to reveal the importance of trophic factors as a therapy for cellular brain repair. The purpose of this study was to analyze whether brain-derived neurotrophic factor (BDNF) administration could mediate oligodendrogenesis and remyelination after white matter injury in subcortical stroke. METHODS: Ischemia was induced in rats by injection of endothelin-1. At 24 hours, 0.4 µg/kg of BDNF or saline was intravenously administered to the treatment and control groups, respectively. Functional evaluation, MRI, and fiber tract integrity on tractography images were analyzed. Proliferation (KI-67) and white matter repair markers (A2B5, 2',3'-cyclic-nucleotide 3'-phosphodiesterase [CNPase], adenomatous polyposis coli [APC], platelet-derived growth factor receptor alpha [PDGFR-α], oligodendrocyte marker O4 [O4], oligodendrocyte transcription factor [Olig-2], and myelin basic protein [MBP]) were analyzed at 7 and 28 days. RESULTS: The BDNF-treated animals showed less functional deficit at 28 days after treatment than the controls (P<0.05). Although T2-MRI did not show differences in lesion size at 7 and 28 days between groups, diffusion tensor imaging tractography analysis revealed significantly better tract connectivity at 28 days in the BDNF group than in the controls (P<0.05). Increased proliferation of oligodendrocyte progenitors was observed in treated animals at 7 days (P<0.05). Finally, the levels of white matter repair markers (A2B5, CNPase, and O4 at 7 days; Olig-2 and MBP at 28 days) were higher in the BDNF group than in the controls (P<0.05). CONCLUSIONS: BDNF administration exerted better functional outcome, oligodendrogenesis, remyelination, and fiber connectivity than controls in rats subjected to subcortical damage in ischemic stroke.

Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function.

This article is a review of cyclic nucleotide phosphodiesterase(s) (CN PDE) from the point of view of the relationships between the newer aspects of the complex enzymology of CN PDE and recent major advances in CN PDE pharmacology. A consolidation of isozyme nomenclature to the proposed family designations is recommended. Emphasis is placed on the importance of defining the subcellular localization of isozymes expressed in a given tissue and cyclic GMP substrate and regulatory roles in CN PDE isozyme functions. CN PDE inhibitors that may be useful for experimental and clinical purposes are discussed. Examples of these inhibitors include CGS 9343B, TCV-3B, KW-6, MIMAX, Dihydroisoquinolines, Trequinsin, bipyridine and dihydropyridazinone cardiotonics, Rolipram, SQ 65442, Zaprinast and Dipyridamole.

Full activation without calmodulin of calmodulin-dependent cyclic nucleotide phosphodiesterase by acidic glycosphingolipids: GM3, sialosylneolactotetraosylceramide and sulfatide.

Among calmodulin-non-binding glycosphingolipids, GM3, sialosylneolactotetraosylceramide (LM1), and sulfatide potently activated calmodulin-dependent cyclic nucleotide phosphodiesterase with or without Ca2+ showing ED50 1-5 microM. In contrast to calmodulin-binding gangliosides, these glycosphingolipids activated the enzyme up to the maximum level achieved by Ca2+/calmodulin and did not inhibit the activity at higher concentrations. Competition studies with GD1b that bind both to calmodulin and the enzyme suggest that the calmodulin-non-binding glycosphingolipids activate the enzyme through interaction with the same site of the enzyme as GD1b interacts.

Effect of flosequinan upon isoenzymes of phosphodiesterase from guinea-pig cardiac and vascular smooth muscle.

The effect of flosequinan and its sulphone metabolite BTS 53,554, on phosphodiesterase isoenzymes isolated from guinea-pig cardiac and vascular smooth muscle using DEAE-cellulose chromatography was investigated. Zaprinast and milrinone showed peak I and peak III selectivity, and IBMX non-selective activity respectively, against both cardiac and vascular smooth muscle isoenzymes, as expected for these reference inhibitors. Flosequinan and BTS 53,554 demonstrated non-selective inhibition with similar potency against both cardiac and vascular smooth muscle isoenzymes and, overall, were the least potent compounds tested. The high inhibitory concentrations observed (IC50 peak III 660 microM for cardiac tissue and 230 microM for vascular smooth muscle with flosequinan) relative to its clinically effective plasma concentration (10 microM) questions the relevance of phosphodiesterase inhibition to the efficacy of flosequinan in heart failure.

Interactions of calmodulin antagonists with calcium antagonists binding sites.

Calmodulin antagonists have calcium entry blocking properties. In order to quantitatively investigate the interactions of these drugs with calcium channels, their effect on [3H]nitrendipine and [3H]d-cis-diltiazem binding to rat cerebral cortex membrane preparations was compared to their inhibitory effect on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The potency of most antagonists to inhibit [3H]nitrendipine binding was correlated with their calmodulin inhibitory potency. However, bepridil (K0.5 = 280 nM), chlorpromazine (K0.5 = 3 microM), triflupromazine (K0.5 = 1.5 microM), imipramine (K0.5 = 3 microM) and propranolol (K0.5 = 14 microM) were much more active on [3H]d-cis-diltiazem binding than on either [3H]nitrendipine binding or calmodulin, suggesting that these compounds bind to higher affinity sites on the calcium antagonist target protein. Moreover, the potencies of these compounds to compete with [3H]d-cis-diltiazem and to inhibit calcium-induced contractions in depolarized smooth muscle were correlated (R = 0.76, p less than 0.02). These data suggest that low concentrations of these hydrophobic drugs which have calcium and calmodulin antagonistic properties inhibit smooth muscle contraction through calcium entry blockade, not calmodulin antagonism.

Identification and characterization of isoenzymes of cyclic nucleotide phosphodiesterase in human kidney and heart, and the effects of new cardiotonic agents on these isoenzymes.

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]- 2 -methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2H)-pyridazinone ) and its metabolites, OPC-18790 ((+/-)-6-[3-(3,4-dimethoxybenzylamino)-2 -hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-1-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone ) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated. PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors. In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDE alpha), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDE beta), which eluted later than cGI-PDE, had been previously isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and morphological effects of fumonisin B(1) on primary cultures of rat cerebrum.

Chronic dietary consumption of the mycotoxin fumonisin B(1) (FB(1)) is associated with leukoencephalomalacia and neuronal degeneration, but identification of the cellular mechanisms underlying this neurotoxicity is difficult due to concurrent adverse systemic changes. For this reason, the present investigation used an in vitro approach to assess the short-term consequences of direct FB(1) (0. 5-75 microM) exposure on astrocytes and oligodendrocytes in primary cultures of rat cerebrum. Beginning at 5 days in vitro, the cultures were exposed to FB(1) at five concentrations (0.5-75 microM), and the cultures were evaluated at 10 and 15 days in vitro. The levels of the sphingolipid-associated constituents sphingosine and sphinganine were determined with a high-performance liquid chromatography. Relative to untreated cultures, exposure to FB(1) diminished the levels of sphingosine at 15 days in vitro, whereas FB(1)-exposed cultures showed significantly increased sphinganine levels and sphinganine/sphingosine ratios. In addition to these changes in sphingolipid constituents, FB(1)-exposed (0.5-75 microM) cultures exhibited a two-fold increase in the number of process-bearing cells by 15 days in vitro. Also, the activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase, an enzyme associated with myelin and oligodendrocytes, was increased in FB(1)-treated cultures. This study suggests that short-term exposure to FB(1) may modify the proliferation or differentiation of glial cells.

[The effect of opioid peptides on a decreased proliferation and on an increased maturation rate of glioma C-6 cells manifested by enhanced marker enzyme activity]. / Vliianie opioidnykh peptidov na snizhenie proliferatsii i uvelichenie skorosti sozrevaniia kletok gliomy C-6, proiavliaemoe povysheniem aktivnosti markirovannykh fermentov.

A study was made of the effect of opioid peptides, (Leu)-enkephalin and its synthetic analog dalargin, on the maturation speed (differentiation) and proliferation activity of glioma C-6 cells. This effect on phenotypes of glioma C-6 cells was determined using some biochemical parameters: changes in the activity of glutamine synthetase (astrocytic marker) and cyclic nucleotide phosphorohydrolase (oligodendrocytic marker) in the culture of glioma C-6 cells in the early and late passages. The biochemical analysis was made at the Laboratory in Denver, USA, and we thank Prof. A. Vernadakis for the possibility to carry out a part of this work that helped us to find the growth activity of opioid peptides on the C-6 cells. Proliferation was examined in cultivation conditions approximately conforming the conditions of cultivation for the primary glial cell cultures. The control proliferation level was high in this case. It is demonstrated that opioid peptides accelerate (or strengthen) the expression of phenotypic signs in C-6 glioma cells in early and late passages changing specific activity of the marker enzymes, i.e. operating as a growth factor. Opioid peptides show glial growth factor characteristics on the glioma C-6 glial cell model as well, for glioma C-6 is known to be a perfect model to analyse the action of different substances on the glia.

Damage to oligodendrocytes in the striatum after MPTP neurotoxicity in mice.

We investigated the alteration of oligodendrocytes in comparison with that of astrocytes and microglia in the mouse striatum after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropridine) treatment under the same conditions using Western blot analysis and Immunohistochemistry. In our Western blot analysis, four administrations of MPTP at 2-h intervals to mice produced the remarkable loss of TH (tyrosine hydroxylase) protein levels in the striatum after 3 and 7 days. In contrast, GFAP (glial fibrillary acidic protein) and Iba-1 protein in the striatum showed a significant increase of GFAP and Iba-1 protein levels 3 and 7 days after MPTP treatment. On the other hand, the levels of CNPase (2', 3'-cyclic nucleotide 3'-phosphodiesterase) protein were decreased significantly in the striatum 3 and 7 days after MPTP treatment. In our immunohistochemical study, a significant decrease in the area of expression of CNPase-positive profiles was observed in the striatum 3 and 7 days after MPTP treatment. These results demonstrate that oligodendrocytes in the striatum are damaged after MPTP treatment. Thus our present findings provide valuable information for the pathogenesis of Parkinson's disease.

Initial biochemical and functional characterization of cyclic nucleotide phosphodiesterase isozymes in canine colonic smooth muscle.

Cyclic nucleotides mediate relaxation of gastrointestinal smooth muscle. The intracellular concentration of these second messengers is determined by a balance between their synthesis and metabolism. Because cyclic nucleotide phosphodiesterase enzymes (PDE) are the sole enzymes responsible for their degradation, it is essential to determine the role of the various PDE isozymes in regulating cyclic nucleotide content of gastrointestinal smooth muscle. To examine the role of different PDE isozymes in colonic smooth muscle motility, soluble PDE activity was measured in fractions obtained from homogenates of canine colon using DEAE sepharose chromatography. PDE activity was determined using [3H]cyclic AMP (cAMP) (1 microM) or [3H]cyclic GMP (cGMP) (1 microM) as a substrate. Results indicated that colonic smooth muscle contains at least two forms of PDE with a high affinity for cGMP. One form was stimulated by calmodulin (type I) and the other was inhibited by low concentrations of zaprinast (type V). In addition, colonic smooth muscle contains at least two isozymes that prefer cAMP as a substrate. One form was inhibited by SB 94120 and cGMP (type III) and the other by rolipram (type IV). An additional peak of PDE activity was identified. The hydrolysis of cAMP by this peak was greatly enhanced by the presence of cGMP, suggesting that this activity belonged to type II or cGMP-stimulated PDE. The functional role of these isozymes was evaluated by determining the ability of selective PDE inhibitors to antagonize a carbachol (0.3 microM)-induced contraction of isolated circular colonic muscle strips in the presence of forskolin (0.2 microM). Concentration-dependent decreases in contractile activity were observed with the following potency order: rolipram > Ro 20-1724 > isobutyl methylxanthine > SB 94120 > zaprinast. These results demonstrate that colonic smooth muscle contains several PDE isozymes and that selective inhibition of PDE isozymes can increase cyclic nucleotide content and antagonize contractile responses. Functionally, PDE IV appears to be very important in reducing contractile activity, suggesting that selective PDE IV inhibitors might be useful in the treatment of gut hypermotility disorders.

Bisphenol A exerts thyroid-hormone-like effects on mouse oligodendrocyte precursor cells.

We report studies on the mechanism of action of bisphenol A (BPA) on the differentiation of oligodendrocyte precursor cells (OPCs). Our results show that: (1) BPA inhibits the differentiation of OPCs induced by exposure to thyroid hormone (T3). (2) The effect is mediated through various mechanisms via the thyroid hormone receptor (TRbeta1) which is considered to be responsible for OPC differentiation. (3) The action of BPA on OPC differentiation does not involve the FcRgamma-Fyn-myelin basic protein (MBP) cascade as an inducer of OPC differentiation nor does it suppress CREB phosphorylation, which is considered to be induced by the T3-TR complex. (4) The presence of MBP isoforms (21.5, 18.5, 17.0 and 14.0 kDa) was detected in OPCs, and the expression of exon 2-containing isoforms (i.e. 17.0 and 21.5 kDa) was upregulated upon treatment with T3. In contrast, expression of MBP was inhibited by BPA.

Dietary polyunsaturated fatty acids modulate fatty acid composition and early activation steps of concanavalin A-stimulated rat thymocytes.

The early biochemical responses to concanavalin A (Con A) of thymocytes from rats fed a saturated (coconut oil), (n-6) (sunflower oil) or (n-3) (fish oil) fatty acid-enriched diet for 3 wk were investigated. Fish oil feeding resulted in greater (n-3) polyunsaturated fatty acid level (PUFA) at the expense of (n-6) PUFA in total and individual thymocyte phospholipids. Such alterations of the fatty acid composition did not affect basal ornithine decarboxylase (ODC), cyclic nucleotide phosphodiesterase (PDE) or gamma-glutamyl transferase activities. However, the fish oil-enriched diet impaired some of the early thymocyte responses to Con A, such as the rapid induction (30 min) of soluble ODC and PDE activities. Synthesis of [3H]20:4(n-6) oxygenated metabolites was not different between the dietary groups; however, the uptake of [3H]20:4(n-6) into phospholipid classes was significantly lower in phosphatidylcholine and greater in phosphatidylethanolamine and phosphatidylinositol after fish oil feeding. Similarly, the Con A-induced remodeling of the [3H]20:4(n-6) esterification in phospholipids differed in sunflower oil- vs. fish oil-fed rats, suggesting a modulation of acyl CoA synthase and/or acyl CoA transferase activities. Thus, the modulation of Con A-induced ODC and PDE stimulation upon in vivo changes of membrane phospholipid fatty acid composition is not related to eicosanoid formation, but rather to the modification of the fatty acid acylation processes, altering phospholipid composition and signal transduction.

Molecular mechanisms involved in the actions of apotransferrin upon the central nervous system: Role of the cytoskeleton and of second messengers.

Apotransferrin (aTf), intracranially administered into newborn rats, produces increased myelination with marked increases in the levels of myelin basic protein (MBP), phospholipids and galactolipids, and mRNAs of MBP and 2', 3' cyclic nucleotide 3'-phosphohydrolase (CNPase). Cytoskeletal proteins such as tubulin, actin, and microtubule-associated proteins are also increased after aTf injection. In contrast, almost no changes are observed in myelin proteolipid protein (PLP) or in its mRNA or cholesterol. In the present study, we used brain-tissue slices and cell cultures highly enriched for oligodendroglia to investigate signaling pathways involved in the action of aTf, and to find out whether cytoskeletal integrity and dynamics were essential for its action upon the neural expression of certain genes. Treatment of brain-tissue slices with aTf produced a marked increase in the expression of MBP, CNPase, and tubulin mRNAs. Colchicine, cytochalasin, and taxol severely reduced the effect of aTf. Addition to cultures of an antibody against transferrin receptor (TfR), protein kinase inhibitors, or a cyclic AMP (cAMP) analogue showed that a functionally intact TfR was necessary, and that tyrosine kinase, protein kinase C and A, as well as calcium-calmodulin-dependent kinase (Ca-CaMK) activities appeared to mediate aTf actions upon the expression of the above mentioned genes. Changes in the levels of phosphoinositides and cAMP induced by aTf in oligodendroglial cell (OLGc) cultures correlated with these results and coincide with an activation of the cyclic response element binding protein (CREB) and of mitogen activated protein kinases. The increased expression of certain myelin genes produced by aTf appear to be mediated by interaction of this glycoprotein with its receptor, by the cytoskeleton of the OLGc, and by a complex activation of protein kinases which lead to CREB phosphorylation.