Carmen Sato-Bigbee

Different Neuroligands and Signal Transduction Pathways Stimulate CREB Phosphorylation at Specific Developmental Stages Along Oligodendrocyte Differentiation

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 β‐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.

Possible Role of CREB in the Stimulation of Oligodendrocyte Precursor Cell Proliferation by Neurotrophin-3

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 β‐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.

Effect of cyclic AMP on the expression of myelin basic protein species and myelin proteolipid protein in committed oligodendrocytes: differential involvement of the transcription factor CREB.

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. J. Neurosci. Res. 66:37–45, 2001.

Neurotrophin-3 and a CREB-mediated signaling pathway regulate Bcl-2 expression in oligodendrocyte progenitor cells

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.

Treatment of oligodendrocytes with antisense deoxyoligonucleotide directed against CREB mRNA: Effect on the cyclic AMP‐dependent induction of myelin basic protein expression

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.

FTY720 Reduces Inflammation and Promotes Functional Recovery after Spinal Cord Injury

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.

Presence of a cyclic AMP response element-binding protein in oligodendrocytes

Abstract: Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocyte 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 cisacting 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 ˜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.

Purification of oligodendrocytes and their progenitors using immunomagnetic separation and Percoll gradient centrifugation.

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.

Recovery of adult oligodendrocytes is preceded by a “lag period” accompanied by upregulation of transcription factors expressed in developing young cells

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 (T3) 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 T3 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.

Schwann cells stimulated by axolemma-enriched fractions express cyclic AMP responsive element binding protein.

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 μg/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.