Jorge N. Larocca

Acetylcholine agonists stimulate mitogen-activated protein kinase in oligodendrocyte progenitors by muscarinic receptors

Oligodendrocytes, the myelin‐producing cells of the central nervous system, express muscarinic acetylcholine receptors (mAChR). Activation of this neurotransmitter receptor by the stable acetylcholine analog carbachol (CCh) triggers transducing events, modulating c‐fos expression and cellular proliferation. To elucidate the signal transduction pathways involved in the transmission of these cellular events, we examined the ability of CCh to activate mitogen‐activated protein kinase (MAPK) in primary cultures of oligodendrocyte progenitors prepared from newborn rat brain. CCh produced a concentration‐ and time‐dependent increase in MAPK activity (predominantly the p42mapk or ERK2) as determined by in‐gel MBP kinase assays. Using the non‐selective muscarinic antagonist atropine we determined that MAPK‐activation by CCH is mediated by muscarinic receptors. In the presence of PD098059, a specific inhibitor of MAPK kinase (MEK), MAPK activity was blocked. Similarly, the presence of extracellular calcium was required for CCh‐mediated MAPK activation. To further elucidate the mechanisms involved in MAPK activation by CCh, the role of PKC was studied. In cells in which protein kinase had been downregulated by chronic treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), the effect of carbachol on MAPK activation was maintained. In contrast, the response to CCh was blocked by the PKC inhibitors H7 and bisindolylmaleimide GF109203X. Our results suggest that MAPK is implicated in the transmission of the signal for mACh receptors and involves a TPA‐insensitive PKC pathway. Further work is required to define the upstream and downstream events which result in CCh‐mediated MAPK activation and proliferation of oligodendrocyte progenitors. J. Neurosci. Res. 50:743–754, 1997.

Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium

Slices of rat brainstem that had been prelabeled by in vivo injection of [3H]inositol were stimulated with carbachol in the presence of lithium and changes measured in the radioactivity of inositol lipids and water-soluble inositol phosphates. For the latter, significant increases were seen for inositol mono- and bisphosphate but not inositol trisphosphate. Analysis of whole tissue phosphoinositides revealed significantly reduced radioactivity in phosphatidylinositol and phosphatidylinositol 4-phosphate, whereas myelin showed decreases in those as well as phosphatidylinositol 4,5-bisphosphate. These effects were blocked by atropine. Stimulation of the tissue slices with elevated K+ resulted in increased formation of inositol phosphate and decreased radioactivity in phosphatidylinositol. The effect was not blocked by atropine and in the presence of this agent, which reduced background reaction, all 3 phosphoinositides showed significant K+-induced loss of label. Elevated K+ and carbachol thus function through different mechanisms in this system. Carbachol is believed to affect myelin phosphoinositides through direct interaction with muscarinic receptors which were recently shown to be present in this membrane.

Isolation of myelin.

The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane. Three protocols are outlined in this unit: isolation of a highly‐purified myelin fraction from the central nervous system (CNS); separation of a highly‐purified CNS myelin fraction into subfractions of different densities; and isolation of myelin from the peripheral nervous system (PNS).

Detection of G Proteins in Purified Bovine Brain Myelin

Abstract: Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]GTPγS) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 ± 9 vs. 271 ± 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5′‐adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [α‐32P]GTP bound to three bands in the 21–27‐kDa range in a manner inhibited by GTP and GTPγS but not App(NH)p. ADP‐ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholatc extract of myelin subjected to chromatography on a column of phenyl‐Sepharose gave at least three major peaks of [35S]GTPγS binding activity. SDS‐PAGE and immunoblot analyses of peak I indicated the presence of Goα, Giα, and Gsα. Further fractionation of peak II by diethyl‐aminoethyl‐Sephacel chromatography gave one [35S]GTPγS binding peak with the low‐molecular‐mass (21–27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS‐PAGE. Immunoblot analysis of this material identified the 36‐kDa protein as the β subunit, whereas the fraction containing 40‐kDa polypeptides reacted with specific antibodies to Goα and Giα. Thus, purified myelin from bovine brain has been shown to contain several GTP‐binding proteins resembling in broad outline the G proteins of whole brain and potentially able to transduce signals received by muscarinic (and perhaps other) receptors in this membrane.

Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors

Oligodendroglial cells express ionotropic glutamate receptors of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA) and kainate (KA) subtypes. Recently, we reported that AMPA receptor agonists increased 45Ca2+ uptake and phospholipase C (PLC) activity. To further elucidate the intracellular signaling mechanisms, we examined the effects of AMPA and KA on mitogen-activated protein kinase (MAPK). KA caused a time- and concentration-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) and the effect was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a competitive AMPA/KA receptor antagonist. Furthermore, the noncompetitive antagonists of AMPA receptor GYKI 52466 and LY 303070 prevented the actions of the agonists, indicating that the effect of KA on MAPK activation is mediated through AMPA receptors in oligodendrocyte progenitors. Chelation of extracellular Ca2+ by EDTA or inhibition of PLC with U73122 abolished MAPK activation by KA. In addition, KA-stimulated MAPK activation was reduced by the protein kinase C (PKC) inhibitors, H7 and bisindolylmaleimide, as well as downregulation of PKC by prolonged exposure to phorbol esters. The involvement of PKC in the signal transduction pathways was further supported by the ability of KA to induce translocation of PKC measured by [3H]PDBu binding. Interestingly, a wortmannin-sensitive phosphatidylinositol 3-kinase and a pertussis toxin (PTX)-sensitive G protein form part of the molecular pathways mediating MAPK activation by AMPA receptor. A specific inhibitor of MAPK kinase, PD 098059, blocked MAPK activation and reduced KA-induced c-fos gene expression. All together, these results indicate that MAPK is implicated in the transmission of AMPA signaling to the nucleus and requires extracellular Ca2+, and PLC/PKC activation.

Vesicle transport in oligodendrocytes: probable role of Rab40c protein.

Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843‐base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP‐binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS) with a Kd of 21 μM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.

Characterization of the signal transduction pathways mediating noradrenaline‐stimulated MAPK activation and c‐fos expression in oligodendrocyte progenitors

In examining the signaling transduction pathway of adrenoceptors in oligodendrocyte progenitors, we have found that stimulation of α1‐adrenoceptors with norepinephrine (NE), in the presence of 3 μM propranolol, increased the activity of mitogen‐activated protein kinases (MAPKs). This stimulation was concentration‐ and time‐dependent, with maximal response after 10 min of exposure to 10 μM NE. Pertussis toxin (PTX) blocked NE‐mediated MAPK activation, suggesting that α1‐adrenoceptor activates MAPK through a PTX‐sensitive G‐protein. In the presence of U73122, an inhibitor of phospholipase C (PLC), MAPK activation was blocked. In oligodendrocyte progenitor cultures, chronic treatment with phorbol‐12‐myristate‐13‐acetate (PMA) down‐regulated protein kinase C (PKC) and blocked NE‐mediated MAPK activation. The response to NE was also significantly decreased by the PKC inhibitors H7 and bisindolylmaleimide GF109203X. Similarly, the effect of NE on MAPK activation was not observed in a calcium‐free medium. Furthermore, attenuation of MAPK activity was observed when cultures were pretreated with LY294002 and wortmannin, inhibitors of phosphatidylinositol‐3 kinase (PI3K). These results suggest that α1‐adrenoceptor‐mediated activation of MAPK involves a PTX‐sensitive G‐protein, PLC, PI3K, and 1,2‐diacyl glycerol (DAG)‐dependent PKC isozyme. Stimulation of oligodendrocyte progenitors with NE also resulted in an increase in c‐fos expression, which was mediated by both α1‐ and β‐adrenoceptor and was calcium‐, PKC‐, and protein kinase A (PKA)‐dependent. Interestingly, in the presence of PD 098059, a specific inhibitor of MAPK kinase (MEK), both MAPK activity and c‐fos expression were blocked. This suggests that MAPK is implicated in the transmission of the signal from α1‐adrenoceptor to c‐fos gene expression. J. Neurosci. Res. 58:765–778, 1999.

Molecular analysis of the monomeric GTP-binding proteins of oligodendrocytes.

Vesicle transport plays an important role in the formation of myelin. Transport of proteins, including proteolipid protein and myelin associated glycoprotein, from their site of synthesis in the endoplasmic reticulum in the perikaryon of the oligodendrocytes, to myelin, takes place via carrier vesicles. The mechanisms that regulate vesicle transport in oligodendrocytes are largely unknown. The presence of monomeric GTP-binding proteins in myelin and oligodendrocytes suggested the hypothesis that these proteins participate in the regulation of vesicle transport. In an attempt to identify the Rab and Rho GTP-binding proteins present in oligodendrocytes, a cDNA library specific for these proteins was generated using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Twelve different clones containing sequences that coded for members of the Rab and Rho families of GTP-binding proteins were isolated. This group includes Rab1, -1b, -2, -5b, -5c, -7, -8, -12, -14, -23 and Rho A. One additional clone revealed a novel cDNA sequence. Analysis of the effector loop motif indicated that this sequence encodes for a member of the Rab family. We refer to this new sequence as Rab0. Comparison of Rab0 with the most similar rat Rab sequences, Rab 14 and Rab 22, and with a recently cloned human Rab22b, showed a 71%, 72% and 94% identity, respectively. By RT-PCR analysis the Rab0 mRNA was found to be mainly expressed in oligodendrocytes and to a lesser extent in oligodendrocyte precursors, astrocytes and microglia. Moreover, the highest levels of Rab0 mRNA were observed in areas of the brain that are heavily myelinated. Rab0 mRNA was also detected in other tissues such as kidney, liver, skeletal muscle. These data provide initial evidence regarding signal transduction pathways that regulate intracellular transport in oligodendrocytes.

Isoprenylated proteins in myelin.

Abstract: Incubation of rat brainstem slices with [3H]‐ mevalonate ([3H]MVA) in the presence of lovastatin resulted in the incorporation of label into three groups of myelin‐associated proteins with molecular masses of 47, 21–27, and 8 kDa, as revealed on sodium dodecyl sulfate‐ polyacrylamide rod gel electrophoresis. Although the gel patterns of [3H]MVA‐derived prenylated proteins were similar, the relative level of 3H incorporated into each protein species differed between myelin and the brainstem homogenate. Immunoprecipitation studies identified the 47‐kDa prenylated protein as a 2′‐3′‐cyclic nucleotide phospho‐ diesterase, whereas the 8‐kDa protein proved to be the γ subunit of membrane‐associated guanine nucleotide regulatory protein. The 3H‐labeled 21–27‐kDa group in myelin corresponds to the molecular mass of the extensive Ras‐ like family of monomeric GTP‐binding proteins known to be prenylated in other tissues. Increase in lovastatin concentration resulted in reduced levels of [3H]MVA‐labeled species in myelin and concomitantly increased levels in the cytosol. A cold MVA chase restored to normality the appearance of [3H]MVA‐labeled proteins in myelin. Furthermore, a high lovastatin concentration in the brainstem slice incubation mixture altered the appearance of newly synthesized nonprenylated myelin proteins, including proteolipid protein and the 17‐kDa subspecies of myelin basic protein. Because other myelin proteins were unaffected by the high lovastatin concentration, restricting the availability of MVA in myelin‐forming cells may selectively alter processes required for myelinogenesis. Although the molecular basis for the” different MVA requirements in myelin‐ forming cells remains undefined, it may involve an alteration in the biological activity of certain proteins that require prenylation to be functionally active, and that are responsible for promoting insertion of specific proteins into the myelin membrane.

Expression and signal transduction of the glucagon receptor in βTC3 cells

The expression and signal transduction of the glucagon receptor (GR) have been studied in betaTC3 cells. Northern blot and RT-PCR analysis indicated the expression of the GR gene in betaTC3 cells. One-5 nM glucagon stimulated a 2.5-fold increase in the IP(S) production. At glucagon concentrations higher than 5 nM, the production of IP(S) was blunted but not abolished. The accumulation of intracellular cAMP was observed following the stimulation with 5 nM of glucagon. A maximal 4.5-fold increase in cAMP was observed using 250 nM glucagon and higher. Comparative studies using a glucagon anatogonist, des-His1[Glu]9glucagon, showed no effect on intracellular cAMP and IPs in betaTC3 cells. Our data shows that the GR gene is expressed in betaTC3 cells. The GR in betaTC3 cells transmits its intracellular signal by causing the accumulation of both IP(S) and cAMP.