Carmen Sato-Bigbee

Virginia Commonwealth University, Richmond, VA, United States

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Publications (22)87.39 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.
    Neurochemical Research 10/2013; · 2.13 Impact Factor
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    ABSTRACT: Although the classical function of myelin is the facilitation of saltatory conduction, this membrane and the oligodendrocytes, the cells that make myelin in the central nervous system (CNS), are now recognized as important regulators of plasticity and remodeling in the developing brain. As such, oligodendrocyte maturation and myelination are among the most vulnerable processes along CNS development. We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. Perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is particularly intriguing because the NOP receptor/nociceptin system has been primarily linked to behavior and pain regulation, but a role in CNS development or myelination has not been described before. Our findings suggest that balance between signaling mediated by (a) MOR activation and (b) a novel, yet unidentified pathway that includes the NOP receptor, plays a crucial role in the timing of oligodendrocyte maturation and myelin synthesis. Moreover, exposure to opioids could disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination.
    Glia 01/2012; 60(1):125-36. · 5.07 Impact Factor
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    ABSTRACT: Stability of the myelin-axon unit is achieved, at least in part, by specialized paranodal junctions comprised of the neuronal heterocomplex of contactin and contactin-associated protein and the myelin protein neurofascin 155. In multiple sclerosis, normal distribution of these proteins is altered, resulting in the loss of the insulating myelin and consequently causing axonal dysfunction. Previously, this laboratory reported that mice lacking the myelin-enriched lipid sulphatide are characterized by a progressive deterioration of the paranodal structure. Here, it is shown that this deterioration is preceded by significant loss of neurofascin 155 clustering at the myelin paranode. Interestingly, prolonged electrophoretic separation revealed the existence of two neurofascin 155 bands, neurofascin 155 high and neurofascin 155 low, which are readily observed following N-linked deglycosylation. Neurofascin 155 high is observed at 7 days of age and reaches peak expression at one month of age, while neurofascin 155 low is first observed at 14 days of age and constantly increases until 5 months of age. Studies using conditional neurofascin knockout mice indicated that neurofascin 155 high and neurofascin 155 low are products of the neurofascin gene and are exclusively expressed by oligodendrocytes within the central nervous system. Neurofascin 155 high is a myelin paranodal protein while the distribution of neurofascin 155 low remains to be determined. While neurofascin 155 high levels are significantly reduced in the sulphatide null mice at 15 days, 30 days and 4 months of age, neurofascin 155 low levels remain unaltered. Although maintained at normal levels, neurofascin 155 low is incapable of preserving paranodal structure, thus indicating that neurofascin 155 high is required for paranodal stability. Additionally, comparisons between neurofascin 155 high and neurofascin 155 low in human samples revealed a significant alteration, specifically in multiple sclerosis plaques.
    Brain 02/2010; 133(Pt 2):389-405. · 9.92 Impact Factor
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    ABSTRACT: A robust and complex inflammatory cascade is known to be a prominent component of secondary injury following spinal cord injury (SCI). Specifically, the concept of trauma-induced autoimmunity has linked the lymphocyte population with neural tissue injury and neurologic deficit. FTY720, a sphingosine receptor modulator that sequesters lymphocytes in secondary lymphoid organs, has been shown to be effective in the treatment of a variety of experimental autoimmune disorders. Accordingly, by reducing lymphocyte infiltration into the spinal cord following SCI, this novel immunomodulator may enhance tissue preservation and functional recovery. In the present study, a moderate to severe contusion SCI was simulated in adult Long-Evans hooded rats. Using flow cytometry we showed that daily FTY720 treatment dramatically reduced T-cell infiltration into the SCI lesion site at 4 and 7 days post-injury, while other inflammatory cell populations were relatively unaltered. To assess functional recovery, three groups of injured animals (treated, vehicle, and injury only) were evaluated weekly for hindlimb recovery. Animals in the treated group consistently exhibited higher functional scores than animals in the control groups after 2 weeks post-injury. This finding was associated with a greater degree of white matter sparing at the lesion epicenter when cords were later sectioned and stained. Furthermore, treated animals were found to exhibit improved bladder function and a reduced incidence of hemorrhagic cystitis compared to control counterparts. Collectively these results demonstrate the neuroprotective potential of FTY720 treatment after experimental SCI.
    Journal of neurotrauma 08/2009; 26(12):2335-44. · 4.25 Impact Factor
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    ABSTRACT: Neurotrophin-3 (NT-3) regulates oligodendrocyte (OLG) differentiation by mechanisms that remain poorly understood. Exposure of OLGs to NT-3 induces a significant increase in the levels of myelin basic protein (MBP). However, we found that this stimulation occurs in the absence of measurable effects on MBP gene promoter activation or mRNA expression, suggesting that NT-3 upregulates MBP protein expression by a posttranscriptional mechanism. Furthermore, NT-3 also causes an increase in the levels of myelin-associated glycoprotein (MAG) and myelin OLG glycoprotein (MOG), raising the possibility of a more general effect on myelin protein synthesis. Surprisingly, (35)S-methionine incorporation into total OLG proteins demonstrated a 50% increase in labeling following only a brief, 15-min treatment with NT-3. Such a remarkably fast response is unlikely due to transcriptional activation, reinforcing the possibility that NT-3 may play a crucial role in regulating protein expression by a posttranscriptional mechanism. In support of this idea, we found that NT-3 stimulates the phosphorylation of essential regulators of the initiation machinery, eukaryotic initiation factor 4E (eIF4E), and its inhibitory binding partner 4E binding protein 1 (4EBP1), two crucial players in controlling cap-dependent protein synthesis. This stimulation involves the activation of pathways mediated by ERK1/2 and PI3K/mTOR, implicating these two kinase systems as modulators of protein synthesis in developing OLGs. Altogether, these observations show for the first time that NT-3 has the capacity of targeting the translational machinery and suggest a potential stimulatory effect of this neurotrophin on myelination by direct action on protein translation in the OLGs.
    Glia 06/2009; 57(16):1754-64. · 5.07 Impact Factor
  • Rochelle P Coelho, Harsimran S Saini, Carmen Sato-Bigbee
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    ABSTRACT: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide variety of biological effects in different cells and tissues. This review discusses the effects of S1P signaling in oligodendrocytes, the myelin making cells of the central nervous system (CNS). Results from different laboratories have uncovered direct actions of S1P at different maturational stages along the oligodendroglial lineage. There is also evidence for the existence in oligodendrocytes of interactions between S1P and signaling by factors which, like neurotrophin-3 (NT-3) and platelet-derived growth factor (PDGF), have profound effects on oligodendrocyte development and myelination. Moreover, S1P signaling in oligodendrocytes may not only play an important role during normal CNS development but also offer new therapeutic avenues to stimulate remyelination in demyelinating diseases like multiple sclerosis.
    Prostaglandins & other lipid mediators 04/2009; 91(3-4):139-44. · 2.42 Impact Factor
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    ABSTRACT: Buprenorphine is a mu-opioid receptor partial agonist and kappa-opioid receptor antagonist currently on trials for the management of pregnant opioid-dependent addicts. However, little is known about the effects of buprenorphine on brain development. Oligodendrocytes express opioid receptors in a developmentally regulated manner and thus, it is logical to hypothesize that perinatal exposure to buprenorphine could affect myelination. To investigate this possibility, pregnant rats were implanted with minipumps to deliver buprenorphine at 0.3 or 1 mg/kg/day. Analysis of their pups at different postnatal ages indicated that exposure to 0.3 mg/kg/day buprenorphine caused an accelerated and significant increase in the brain expression of all myelin basic protein (MBP) splicing isoforms. In contrast, treatment with the higher dose caused a developmental delay in MBP expression. Examination of corpus callosum at 26-days of age indicated that both buprenorphine doses cause a significant increase in the caliber of the myelinated axons. Surprisingly, these axons have a disproportionately thinner myelin sheath, suggesting alterations at the level of axon-glial interactions. Analysis of myelin associated glycoprotein (MAG) expression and glycosylation indicated that this molecule may play a crucial role in mediating these effects. Co-immunoprecipitation studies also suggested a mechanism involving a MAG-dependent activation of the Src-family tyrosine kinase Fyn. These results support the idea that opioid signaling plays an important role in regulating myelination in vivo and stress the need for further studies investigating potential effects of perinatal buprenorphine exposure on brain development.
    Glia 08/2008; 56(9):1017-27. · 5.07 Impact Factor
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    ABSTRACT: The immunomodulator 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (FTY720) has promising therapeutic effects in multiple sclerosis (MS), a degenerative disease in which demyelination of the central nervous system is accompanied by death of oligodendrocytes (OLGs), the myelin-producing cells. In vivo phosphorylation of FTY720 generates an agonist for G protein-coupled receptors for sphingosine-1-phosphate, a lipid mediator that plays a crucial role in the stimulation of OLG survival by neurotrophin-3 (NT-3). The mechanisms underlying the action of FTY720 in MS are not clearly understood, although the effects of this drug in autoimmune diseases are thought to stem from its ability to reduce lymphocyte infiltration and inflammation. Interestingly, we now found that FTY720 also has a direct effect on OLG progenitors. Treatment of these cells with FTY720 causes activation of extracellular signal-regulated kinase 1/2 and Akt, accompanied by protection from apoptosis. However, FTY720 also arrested OLG differentiation. Importantly, this effect was counteracted by NT-3, which not only enhanced the survival of OLG progenitors induced by FTY720 but also stimulated their maturation. Altogether, these observations suggest that in addition to its immunosuppressive functions, FTY720 could also have a beneficial effect in MS by direct action on OLG progenitors. However, the finding that FTY720 blocks the differentiation of these cells raises the question of whether MS therapies with FTY720 should include the use of differentiation-enhancing factors such as NT-3. This approach would ensure both protection of existing OLG progenitor pools against immune-mediated insults as well as stimulation of remyelination by enhancing the maturation of these cells.
    Journal of Pharmacology and Experimental Therapeutics 12/2007; 323(2):626-35. · 3.89 Impact Factor
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    ABSTRACT: We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.
    Journal of Neurochemistry 01/2006; 95(5):1298-310. · 3.97 Impact Factor
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    ABSTRACT: Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.
    Journal of Neurochemistry 06/2004; 89(4):951-61. · 3.97 Impact Factor
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    ABSTRACT: Diverse molecular mechanisms have been discovered that mediate the loss of responses to the deprived eye during monocular deprivation. cAMP/Ca2+ response element-binding protein (CREB) function, in particular, is thought to be essential for ocular dominance plasticity during monocular deprivation. In contrast, we have very little information concerning the molecular mechanisms of recovery from the effects of monocular deprivation, even though this information is highly relevant for understanding cortical plasticity. To test the involvement of CREB activation in recovery of responses to the deprived eye, we used herpes simplex virus (HSV) to express in the primary visual cortex a dominant-negative form of CREB (HSV-mCREB) containing a single point mutation that prevents its activation. This mutant was used to suppress CREB function intracortically during the period when normal vision was restored in two protocols for recovery from monocular deprivation: reverse deprivation and binocular vision. In the reverse deprivation model, inhibition of CREB function prevented loss of responses to the newly deprived eye but did not prevent simultaneous recovery of responses to the previously deprived eye. Full recovery of cortical binocularity after restoration of binocular vision was similarly unaffected by HSV-mCREB treatment. The HSV-mCREB injections produced strong suppression of CREB function in the visual cortex, as ascertained by both DNA binding assays and immunoblot analysis showing a decrease in the expression of the transcription factor C/EBPbeta, which is regulated by CREB. These results show a mechanistic dichotomy between loss and recovery of neural function in visual cortex; CREB function is essential for loss but not for recovery of deprived eye responses.
    Journal of Neuroscience 11/2002; 22(20):9015-23. · 6.91 Impact Factor
  • Fatemah S Afshari, Annie K Chu, Carmen Sato-Bigbee
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    ABSTRACT: Cell cultures prepared from oligodendrocytes directly obtained from adult rat brain are composed of mature cells that lose their cell processes and myelin membrane during their isolation and therefore represent a very useful model to investigate the factors that could stimulate their recovery. We have observed that mature oligodendrocytes isolated from adult animals remain as round cells that lack processes for the first 3-4 days in culture. At the end of this lag period, however, the majority of the adult oligodendrocytes show a remarkable recovery, rapidly growing complex and extensive cell processes. Interestingly, the end of this lag period is accompanied by a dramatic upregulation in the expression of thyroid hormone (T(3)) receptor (TR). The functional importance of this increase in TR levels is supported by the observation that the majority of the cells cultured in the presence of T(3) show significantly more extensive and complex process outgrowth than the control cells in cultures lacking this hormone. In addition, this reactivation of the adult cells was also preceded by an increased expression of glucocorticoid receptor (GR) and cyclic AMP-response element binding protein (CREB), two transcription factors that together with TR appear to play important roles in the control of neonatal oligodendrocyte development. Thus, it is possible to hypothesize that upregulation of these proteins may be part of the metabolic changes that occur during the lag period required for recovery of the adult oligodendrocytes. These observations raise the question of whether these transcription factors may play any significant role during remyelination after demyelinating lesions of adult CNS.
    Journal of Neuroscience Research 02/2002; 67(2):174-84. · 2.97 Impact Factor
  • F S Afshari, A K Chu, C Sato-Bigbee
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    ABSTRACT: Our previous results support the idea that CREB (cyclic AMP-response element binding protein) may be a mediator of neuroligand and growth factor signals that, coupled to different signal transduction pathways, play different roles at specific stages of oligodendrocyte development. In the early stages, when cells are immature precursors, CREB may play a role as a mediator of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) pathways regulating cell proliferation. In contrast, at a later stage, when cells are already committed oligodendrocytes, CREB seems to play an important role as a mediator in the stimulation of myelin basic protein (MBP) expression by cyclic AMP (cAMP). In this study, we have investigated whether cAMP and CREB play a role in regulating the expression of all or on the other hand particular MBP isoforms. The results indicated that treatment of committed oligodendrocytes with the cAMP analogue db-cAMP results in a pattern of expression of MBP-related polypeptides that most closely resembles the pattern of MBPs observed in cerebra from adult animals. Experiments in which CREB expression was inhibited using a CREB antisense oligonucleotide, suggested that CREB is involved in the cAMP-dependent stimulation of all the MBP isoforms. In contrast, we have found that db-cAMP stimulates the expression of myelin proteolipid protein (PLP) in a process that occurs despite inhibition of CREB expression. These results support the idea that cAMP stimulates the maturation of oligodendrocytes and stress the fact multiple mechanisms may convey the action of this second messenger modulating oligodendrocyte differentiation and myelination.
    Journal of Neuroscience Research 11/2001; 66(1):37-45. · 2.97 Impact Factor
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    R J Colello, C Sato-Bigbee
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    ABSTRACT: In this unit, two techniques are described for the purification of oligodendrocytes and their progenitors from the developing mammalian central nervous system (CNS). The first method utilizes the technique of immunomagnetic separation to selectively isolate oligodendrocytes and their progenitor cells from the optic nerve of prenatal and early postnatal rats. This technique takes advantage of the surface antigens expressed on these cells. A paramagnetic bead is attached to the cells via an antibody bridge. Target cells that are coupled to magnetic beads can then be separated from a heterogeneous cell population using a magnetic field. The second method for isolating oligodendrocytes uses Percoll gradient centrifugation to separate oligodendrocytes from a heterogeneous cell population by virtue of their cell density and allows the direct isolation of oligodendrocytes from animals aged postnatal day 4 (P-4) to adult. This method is particularly useful for assessing physiological systems present in development that may be lost as a result of growing purified neonatal cells in vitro in the absence of neuronal influence.
    Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.] 06/2001; Chapter 3:Unit 3.12.
  • J R Johnson, A K Chu, C Sato-Bigbee
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    ABSTRACT: We have previously shown that the transcription factor CREB (cyclic AMP-response element binding protein) could be a mediator of neuronal signals that, coupled to different signal transduction pathways, may play different regulatory roles at specific stages of oligodendrocyte (OLG) development. We have found before that in committed OLGs, CREB activation by phosphorylation can be triggered by beta-adrenergic stimulation and appears to play a role in the induction of OLG differentiation by cyclic AMP. In contrast, in OLG precursor cells, CREB phosphorylation is stimulated by neuroligands that increase calcium levels by a process that involves a mitogen-activated protein kinase (MAPK)/protein kinase C (PKC) pathway. This observation suggested that at this early developmental stage, CREB could play a role in regulating cell proliferation. In support of this hypothesis, we have now found that a rapid and dramatic stimulation of CREB phosphorylation is one of the earliest events that precedes the increase in cell proliferation that is observed when OLG precursors are treated with neurotrophin-3 (NT-3). Experiments in which CREB phosphorylation was investigated in the presence of different kinase inhibitors indicated that the activation of this transcription factor in the presence of NT-3 is mediated by the concerted action of MAPK- and PKC-dependent signal transduction pathways. Moreover, our present results also showed that down-regulation of CREB expression in the OLG precursors abolished the increase in DNA synthesis that is observed when the cultures are treated with NT-3. Thus, these results support the idea that in immature OLG precursors, CREB plays an important role in transducing signals which, like NT-3, may regulate cell proliferation.
    Journal of Neurochemistry 05/2000; 74(4):1409-17. · 3.97 Impact Factor
  • C Sato-Bigbee, S Pal, A K Chu
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    ABSTRACT: We have shown previously that the pattern of expression of the transcription factor CREB (cyclic AMP-response element binding protein) in developing oligodendrocytes (OLGs) suggests a role during a period that precedes the peak of myelination in rat brain. We have now investigated the signaling pathways that could be responsible for activating CREB by phosphorylation at different stages along OLG maturation. CREB phosphorylation was studied in short-term cultures of immature OLG precursor cells and young OLGs isolated from 4- and 11-day-old rat cerebrum, respectively. The results indicated that at both developmental stages, CREB phosphorylation could be stimulated by either increased concentrations of cyclic AMP and cyclic AMP-dependent protein kinase activation or increased Ca2+ levels and a protein kinase C activity. The results also showed that CREB phosphorylation in immature OLG precursor cells could be up-regulated by treatment with histamine, carbachol, glutamate, and ATP (neuroligands known to increase Ca2+ levels in these cells), by signaling cascade(s) that involve a protein kinase C activity, as well as the mitogen-activated protein kinase pathway. In contrast, in cells isolated from 11-day-old rats, at a developmental stage that immediately precedes the beginning of the active period of myelin synthesis, CREB phosphorylation was only stimulated by treatment with the beta-adrenergic agonist isoproterenol in a process that appears to be mediated by a cyclic AMP/cyclic AMP-dependent protein kinase-dependent pathway. These results support the idea that CREB could be a mediator of neuronal signals that, coupled to specific signal transduction cascades, may play different regulatory roles at specific stages along OLG differentiation.
    Journal of Neurochemistry 02/1999; 72(1):139-47. · 3.97 Impact Factor
  • C Sato-Bigbee, G H DeVries
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    ABSTRACT: We have shown previously that in oligodendrocytes, the transcription factor cyclic AMP response element binding protein (CREB) is maximally expressed immediately prior to the most rapid period of myelination in rat brain. We have begun to investigate the role of this protein during myelination by downregulating CREB synthesis in cultured oligodendrocytes using an antisense deoxyoligonucleotide directed against CREB mRNA. Neonatal oligodendrocytes were grown for 4 days in a chemically defined medium (CDM) after which intracellular delivery of CREB antisense oligonucleotide was facilitated by using a liposome preparation. Control cultures were treated in a similar manner but in the presence of CREB sense oligomer. Immediately after transfection, cells were cultured for 3 days in CDM in the presence or absence of the cyclic AMP (cAMP) analogue N6, O21-dibutyryl cAMP (db-cAMP). In these cultures, myelin basic protein (MBP) expression was investigated by immunocytochemistry and Western blot analysis. Treatment of control cultures with db-cAMP resulted in a significant increase in the number of MBP positive cells which was abolished when the cells were treated with CREB antisense oligonucleotide. MBP positive cells in control cultures treated with db-cAMP have extended and highly branched MBP positive processes. In contrast, MBP positive cells in either control cultures grown in the absence of db-cAMP or cultures grown in the presence of db-cAMP but treated with CREB antisense oligonucleotide showed shorter and less complex processes and the MBP immunoreactivity appeared to be concentrated in the cell body. These observations suggest that CREB is at least one of the mediators in the induction of oligodendrocyte differentiation by cAMP.
    Journal of Neuroscience Research 11/1996; 46(1):98-107. · 2.97 Impact Factor
  • M M Lee, C Sato-Bigbee, G H De Vries
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    ABSTRACT: Both axolemma-enriched fractions (AEF) and cyclic AMP have been shown to regulate the proliferation and differentiation of cultured primary Schwann cells (SC). We have evaluated the role of CREB, a transcription factor that binds to the cAMP-responsive element, in mediating the AEF-stimulated SC proliferation and differentiation. We detected CREB in nuclear extracts derived from SC stimulated with 40 micrograms/ml of AEF for 16, 24, 48, 72, and 96 hr, using a DNA-electrophoretic mobility shift assay. Unstimulated quiescent SC contained low levels of CREB which increased to a maximal level after 48 hr of AEF treatment. Using anti-CREB antibodies and Western blot analysis, after 24 hr of AEF treatment we first detected CREB as a 45 kDa protein which reached a maximal level of expression after 72 hr. Double labeled immunocytochemistry using anti-CREB and anti-5-bromo-2'-deoxy-uridine antibodies demonstrated maximal CREB expression after 72 hr of AEF treatment, closely coinciding with the temporal expression of SC proliferation. At all times examined, all AEF-treated SC labeled by anti-CREB antibodies were also labeled with anti-BrdU antibodies. These observations are consistent with the view that CREB could play an important role in the induction of SC proliferation by AEF.
    Journal of Neuroscience Research 11/1996; 46(2):204-10. · 2.97 Impact Factor
  • C Sato-Bigbee, E L Chan, R K Yu
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    ABSTRACT: Several laboratories have shown that cyclic AMP (cAMP) plays an important role in inducing oligodendrocyte differentiation and myelin synthesis. Our previous results have shown that oligodendrocytes contain a nuclear protein that binds to the DNA sequence TGACGTCA or cAMP response element (CRE) known to be involved in the transcriptional regulation of cAMP-responsive genes. In this report the oligodendroglial CRE-binding protein was further identified by using two different antibodies which specifically recognize the CRE-binding protein known as CREB. In DNA-shift assays CREB-1(X-12) antibody interacted with the CRE-protein complexes resulting in further retardation ("super shift") of the mobility of the bands in the gels. Immunoprecipitation of oligodendroglial nuclear extracts with CREB(240) antibody prior to the DNA binding assays resulted in a lack of formation of CRE-protein complexes. In addition immunoreaction with CREB(240) antibody identified the CRE-binding species as a 45 kDa phosphoprotein. Immunocytochemical staining with CREB(240) antibody in oligodendrocytes from 10-, 14-, and 18-day-old and adult rats indicated that this protein is expressed before the appearance of myelin basic protein (MBP) which was used as a marker of myelin synthesis. Collectively, these observations support our previous results and indicate that the oligodendroglial CRE-binding protein species is highly homologous to the CREB protein. The developmental expression of this CREB protein supports the idea of a possible role during the early stages of oligodendrocyte differentiation preceding the peak of myelin synthesis in rat CNS.
    Journal of Neuroscience Research 09/1994; 38(6):621-8. · 2.97 Impact Factor
  • C Sato-Bigbee, R K Yu
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    ABSTRACT: Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocytes differentiation. However, the mechanism(s) of this stimulation remains unknown. Because in several cell types the transcriptional activity of various cAMP-responsive genes is regulated through a cis-acting DNA sequence known as cAMP response element (CRE), we investigated the possible presence of a CRE binding (CREB) protein in myelinating oligodendrocytes. A double-stranded oligonucleotide containing a tandem repeat of the CRE sequence was labeled with T4 kinase in the presence of [32P]ATP and then incubated with a nuclear protein extract from 14-day-old rat brain oligodendrocytes. The reaction mixture was then electrophoresed on nondenaturing polyacrylamide gels. The results indicated the presence of a protein that specifically binds to the CRE sequence. The results were supported by southwestern blotting assays in which the CRE probe bound to a approximately 45-kDa protein species. In separate experiments, it was shown that the 45-kDa protein can be phosphorylated in vitro by the catalytic subunit of protein kinase A. Developmental analysis of CREB protein expression indicated a peak at 14 days of age, preceding the peak of myelinogenesis.
    Journal of Neurochemistry 07/1993; 60(6):2106-10. · 3.97 Impact Factor