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Keywords:

  • Myelin basic protein;
  • Mitogen-activated protein kinase;
  • Phosphorylation;
  • Hippocampus;
  • Alveus;
  • Action potential;
  • Reactive oxygen species;
  • Reactive nitrogen species

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Abstract : Myelin basic protein (MBP) phosphorylation is a complex regulatory process that modulates the contribution of MBP to the stability of the myelin sheath. Recent research has demonstrated the modulation of MBP phosphorylation by mitogen-activated protein kinase (MAPK) during myelinogenesis and in the demyelinating disease multiple sclerosis. Here we investigated the physiological regulation of MBP phosphorylation by MAPK during neuronal activity in the alveus, the myelinated output fibers of the hippocampus. Using a phosphospecific antibody that recognizes the predominant MAPK phosphorylation site in MBP, Thr95, we found that MBP phosphorylation is regulated by high-frequency stimulation but not low-frequency stimulation of the alveus. This change was blocked by application of tetrodotoxin, indicating that action potential propagation in axons is required. It is interesting that the change in MBP phosphorylation was attenuated by the reactive oxygen species scavengers superoxide dismutase and catalase and the nitric oxide synthase inhibitor N-nitro-L-arginine. Removal of extracellular calcium also blocked the changes in MBP phosphorylation. Thus, we propose that during periods of increased neuronal activity, calcium activates axonal nitric oxide synthase, which generates the intercellular messengers nitric oxide and superoxide and regulates the phosphorylation state of MBP by MAPK

Myelin basic proteins (MBPs) are a family of positively charged proteins that contribute to the formation and compaction of the myelin sheath. In the CNS, MBPs are found in myelinating oligodendrocytes at the major dense line, the cytoplasmic interface of the myelin sheath (Monuki and Lemke, 1995). MBP is absolutely required for formation of myelin and the major dense line, as a large deletion of the MBP gene in the mutant mouse, shiverer, results in an almost complete absence of myelin in the CNS of these mice (Roach et al., 1983). Moreover, increasing the expression of MBP in shiverer mice restores the major dense line and the myelin sheath in a dose-dependent manner (Shine et al., 1992). It is thought that MBP contributes to the formation of myelin by interactions with proteolipid protein (Edwards et al., 1989) in conjunction with its own homophilic interactions (Smith, 1982). Because MBP is highly positively charged and closely apposed to the negative lipid bilayer of myelin, regulation of the charge nature of MBP could alter the compaction and structure of the myelin sheath.

One such posttranslational modification that alters the charge properties of MBP is phosphorylation. MBP is an excellent substrate for several protein kinases, including protein kinase C, protein kinase A, calcium/calmodulin-dependent protein kinase II, and mitogen-activated protein kinase (MAPK) (reviewed by Ulmer, 1988). Phosphorylation decreases the ability of MBP to aggregate lipid vesicles, increase vesicle permeability, and organize lipids into multilayers (Brady et al., 1985 ; Cheifetz and Moscarello, 1985 ; Cheifetz et al., 1985). These findings suggest that MBP phosphorylation could destabilize the MBP-lipid interactions in the myelin sheath and alter the structure of the myelin sheath.

The regulation of MBP phosphorylation in vivo has yet to be completely understood. In the developing nervous system, MBP phosphorylation occurs immediately before and during myelinogenesis (Ulmer and Braun, 1983 ; Vartanian et al., 1986 ; Stariha et al., 1997). This phosphorylation event is mediated by protein kinase C and MAPK and inhibited by protein kinase A activation. In the optic nerve preparation, depolarization of oligodendrocytes and high-frequency action potential propagation regulate MBP phosphorylation by protein kinase C ; however, other protein kinases have yet to be tested (Murray and Steck, 1983, 1984). Recently, we have found that the phosphorylation state of MBP increases during hippocampal long-term potentiation (Atkins et al., 1997). These studies suggest that regulation of MBP phosphorylation is quite complex, potentially altering the association of MBP with the myelin membrane in a dynamic manner.

Recently, a phosphospecific antibody has been developed against MBP, recognizing the MAPK phosphorylation site, Thr95 (Yon et al., 1995). This site is the predominant in vivo phosphorylation site for MAPK (Erickson et al., 1990 ; Sanghera et al., 1990), and phosphorylation of this site is regulated during myelinogenesis and multiple sclerosis (Yon et al., 1996 ; Stariha et al., 1997). This antibody allows us to assay directly for changes in MBP phosphorylation by MAPK and to determine the regulatory mechanisms for phosphorylation of this site. With this antibody, we investigated the forms of neuronal activity that regulate MBP phosphorylation in the adult nervous system.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Hippocampal slice preparation

Standard rat hippocampal slices were prepared as described (Roberson and Sweatt, 1996). All experiments were performed in compliance with the Baylor College of Medicine Institutional Animal Care and Use Committee and national regulations and policies. Hippocampal slices were perfused in saline solution (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2, saturated with 95% O2/5% CO2, pH 7.4) at 32°C. Drugs were delivered to the hippocampal slices by perfusion in the recording saline. The fast synaptic transmission blockers 6-cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (20 μM), DL-2-amino-5-phosphonovalerate (50 μM), picrotoxin (20 μM), and bicuculline (20 μM) were applied in all experiments to isolate the effects of action potential propagation on MBP phosphorylation. When calcium was omitted from the recording saline, we increased the magnesium concentration to 6 mM. To measure action potential responses generated in the alveus, we stimulated the alveus antidromically, recorded extracellularly in stratum pyramidale of area CA1, and measured the amplitude of the population fiber volley. Low-frequency stimulation (LFS ; every 20 s) was delivered at a stimulus intensity 30-40% of the maximal response. High-frequency stimulation (HFS) was delivered as three sets of tetani (two 100-Hz, 1-s-long tetani, separated by 20 s) separated by 5 min given at the minimal stimulation intensity that elicited 75% of the maximal response. Experimental slices were compared with paired control slices from the same recording chamber that received three test stimulations to ensure viability. CA1 subregions were assayed 45 min posttetanus, after freezing the hippocampal slices on dry ice and microdissecting the CA1 subregions.

Western blotting

CA1 subregions were briefly sonicated in 100 μl of buffer [20 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA, 1 mM Na4P2O7, 100 ng/ml aprotinin, 100 ng/ml leupeptin, and 100 μM phenylmethylsulfonyl fluoride] and boiled with sample buffer. Proteins were separated by 15% polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidine difluoride membranes (Immobilon P). Membranes were blocked in Tris-buffered saline containing Tween 20 with 3% bovine serum albumin. The following primary antibodies were used : phospho-MBP (1 : 2,000, from M. Yon and N. Groome), total MBP (1 : 1,000, from Chemicon ; or 1 : 2,000, from Boehringer Mannheim), and total MAPK (1 : 1,000, from Upstate Biotechnology). The secondary antibodies used were either anti-rabbit conjugated to horseradish peroxidase or anti-mouse conjugated to horseradish peroxidase. Immunoreactivity was detected using the enhanced chemiluminescence method (Amersham). Total MBP amounts for each sample were normalized to total protein amounts using p44 MAPK immunoreactivity or a Folin phenol reagent assay. To assess for changes in MBP phosphorylation levels, immunoreactivity with the phosphospecific MBP antibody was normalized to total MBP amounts and then to total protein amounts in each sample. Densitometry analysis was performed using NIH Image software. Western blots were developed to be linear in the range used for densitometry.

Data analysis

Statistical analysis was conducted by one-way ANOVA using Tukey’s test or Student’s t test. All data are mean ± SEM values.

Phosphorylation of MBP Thr95 is regulated by neuronal activity

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

To investigate regulation of the phosphorylation state of MBP during periods of increased neuronal activity, we studied the alveus, the myelinated CA1 axons in the hippocampus. We chose to study the alveus in the hippocampal slice preparation because it is easily amenable to electrophysiological and pharmacological manipulations. Furthermore, the alveus is the final output pathway of the hippocampus, a structure intensely studied for its critical role in information processing (reviewed by Milner et al., 1998). We delivered electrical stimulation to the alveus and monitored the population spike by extracellular recordings in the stratum pyramidale of area CA1 (Fig. 1A). Both LFS and HFS paradigms were tested to determine the physiological paradigms that regulate MBP phosphorylation. Using an antibody that detected phospho-Thr95, we found that MBP phosphorylation by MAPK was decreased 45 min after delivery of HFS but not LFS (Fig. 1B). There was no significant change in MBP amounts with either HFS or LFS (Fig. 1C), indicating that the change in MBP phosphorylation by HFS could not be accounted for by changes in protein amounts. These results suggest that the MAPK phosphorylation site, Thr95, is regulated by high-frequency action potential firing.

image

Figure 1. MBP is regulated by phosphorylation at Thr95 by highfrequency action potential propagation along CA1 axons. A : HFS (arrows) was delivered to the alveus, and the population spike was monitored in the stratum pyramidale of the CA1 region for 45 min. A representative electrophysiology experiment is shown. Inset : The corresponding physiology traces before (a) and after (b) tetanization. Calibration scale = 2 ms and 4 mV. B : MBP phosphorylation levels decreased following tetanization of the alveus, as assessed by western blotting with a phosphospecific MBP antibody that detects phosphorylated Thr95. A representative western blot and the corresponding densitometric analysis are shown for hippocampal slices that received HFS of the alveus (n = 13, p < 0.05 by ANOVA) as compared with paired control hippocampal slices (CTL ; n = 13). No change in phosphorylation was observed with LFS (n = 14). C : MBP amounts did not significantly increase after HFS (116 ± 11%, n = 12) or LFS (113 ± 5%, n = 14) of the alveus.

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Time course of the change in MBP phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

To investigate the kinetics of the change in MBP phosphorylation following HFS of the alveus, we as- sayed hippocampal slices 5 and 25 min after tetanization for changes in MBP phosphorylation at Thr95. At both time points, there was no significant difference in MBP phosphorylation, although a trend toward decreased phosphorylation was observed (Fig. 2). There was no significant change in MBP amounts at either time point. This result suggests that high-frequency action potential propagation along axons results in a persistent biochemical change in oligodendrocytes that may be subserved by a slowly developing biochemical mechanism, in that it takes a significant amount of time to achieve decreased phosphorylation of MBP.

image

Figure 2. Regulation of MBP phosphorylation at Thr95 is a delayed change in response to high-frequency action potential firing. MBP phosphorylation at Thr95 was assayed by western blotting at 5, 25, or 45 min after HFS of the alveus. Although a small decrease in MBP phosphorylation was observed at 5 (n = 12) and 25 min (n = 13) posttetanus, this decrease was not statistically significant until 45 min (n = 13, **p < 0.01 by ANOVA) after tetanization. No significant changes in MBP amounts were observed at either 5 (114 ± 11%, n = 12) or 25 min (101 ± 6%, n = 13) after HFS of the alveus.

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Action potentials are necessary to regulate MBP phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Stimulating the alveus could potentially regulate MBP phosphorylation by direct electrical activation of the oligodendrocytes ; alternatively, action potential generation in axons could be required. To distinguish between these possibilities, we repeated the preceding experiment in the presence of the sodium channel blocker tetrodotoxin (TTX), which blocks action potential generation in the axons but leaves direct stimulation of the oligodendrocytes intact (Sontheimer, 1994). When HFS was delivered to the alveus in the presence of TTX, the change in MBP phosphorylation was blocked (Fig. 3 ; compare Fig. 1B), indicating that the regulation of MBP phosphorylation in the alveus requires axonal action potential generation.

image

Figure 3. The decrease in MBP phosphorylation at Thr95 requires action potentials elicited in CA1 axons. A : A representative western blot is shown, demonstrating no change in MBP phosphorylation in TTX-treated (500 nM) hippocampal slices given HFS in the alveus as compared with paired control slices (CTL). B : Densitometric analysis confirmed this result (n = 10), indicating that action potential generation in CA1 axons is required for regulation of MBP phosphorylation. There was no change in MBP amounts in HFS slices treated with TTX (91 ± 8%, n = 11).

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All MBP isoforms are regulated by phosphorylation during neuronal activity

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The classical MBP isoforms exist as a family of proteins 14, 17, 18.5, and 21.5 kDa in size (reviewed by Monuki and Lemke, 1995). We determined which MBP isoforms are regulated during high-frequency action potential firing by densitometric analysis of each isoform. We observed a significant reduction in phosphorylation levels of all four isoforms (Fig. 4). This result supports the assertion that the biochemical changes we are observing are occurring in oligodendrocytes, as each of these four oligodendrocyte markers is regulated by phosphorylation during periods of increased neuronal activity. In all further experiments, we analyzed changes in the 18.5-kDa isoform.

image

Figure 4. All four isoforms of MBP (14, 17, 18.5, and 21.5 kDa, denoted by arrows) are regulated by phosphorylation during increased firing of myelinated CA1 axons. The anomalous migration of MBP is due to the highly positively charged nature of the protein and has been reported previously (Campagnoni and Magno, 1974). A : A representative western blot is shown, demonstrating detection of all four MBP isoforms. B : Densitometric analysis of these four MBP isoforms, when assayed for phospho-Thr95 levels by western blotting, indicates that all four isoforms were regulated in hippocampal slices that received HFS of the alveus as compared with paired control slices (CTL) (14 kDa, n = 11, p < 0.01 ; 17 kDa, n = 11, p < 0.001 ; 18.5 kDa, n = 13, p < 0.001 ; 21.5 kDa, n = 11, p < 0.001 by ANOVA).

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Reactive oxygen species mediate neuron-glia signaling

The signaling molecule mediating the communication between neurons and oligodendrocytes in the hippocampus could be one of several possibilities, such as increased extracellular K+ or a diffusible messenger such as nitric oxide, superoxide, or arachidonic acid. We chose to test the hypothesis that the intercellular messenger was a reactive oxygen or nitrogen species for several reasons. First, reactive nitrogen species are generated as intercellular messengers during increased firing of neurons in hippocampal long-term potentiation (Chetkovich et al., 1993 ; Schuman and Madison, 1994). Second, MAPK activation is regulated by reactive oxygen and nitrogen species in hippocampal slices (Kanterewicz et al., 1998) and in vitro (Fialkow et al., 1994 ; Stevenson et al., 1994). Third, we have previously found that a reactive oxygen or nitrogen species is responsible for regulation of MBP phosphorylation via protein kinase C during periods of increased neuronal activity in the hippocampus (C.M.A. and J.D.S., unpublished data). To test this hypothesis, we applied reactive oxygen species scavengers and a reactive nitrogen species inhibitor to hippocampal slices, delivered HFS to the alveus, and then assayed the CA1 subregion for changes in MBP phosphorylation at Thr95 (Fig. 5A). We observed a block of the decreased MBP phosphorylation with application of a superoxide scavenger [superoxide dismutase (SOD)], in conjunction with a hydrogen peroxide scavenger (catalase) and a nitric oxide synthase inhibitor N-nitro-L-arginine (L-NOArg) (Fig. 5B ; compare Fig. 1B). These experiments indicate that superoxide, hydrogen peroxide, nitric oxide, or all three together may be the intercellular messengers between neurons and oligodendrocytes that regulates MBP phosphorylation.

image

Figure 5. Reactive oxygen and nitrogen species signal from CA1 axons to oligodendrocytes and regulate the change in MBP phosphorylation during high-frequency action potential firing. A : SOD (120 units/ml), catalase (260 units/ml), and L-NOArg (50 μM) were applied in the recording saline throughout the experiment. After 16 min of baseline recording, the alveus was tetanized (arrows), and then 45 min after the final tetanus, the CA1 subregion was assayed for changes in MBP phosphorylation. A representative electrophysiology experiment is shown. Inset : Traces were obtained before (a) and after (b) tetanization. Calibration scale = 2 ms and 4 mV. B : Representative western blot and densitometric analysis of paired control (CTL) and HFS slices demonstrates an attenuation of the change in MBP phosphorylation when reactive oxygen species scavengers and a reactive nitrogen species inhibitor were applied (n = 6). C : No change in MBP amount was observed as shown by a representative western blot and densitometric analysis (n = 6).

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To determine if each of these reactive oxygen or nitrogen species contributes to the neuron-glia signaling serving are occurring in oligodendrocytes, as each of these four oligodendrocyte markers is regulated by phosphorylation during periods of increased neuronal activity. In all further experiments, we analyzed changes in the 18.5-kDa isoform.

Reactive oxygen species mediate neuron-glia signaling

The signaling molecule mediating the communication between neurons and oligodendrocytes in the hippocampus could be one of several possibilities, such as increased extracellular K+ or a diffusible messenger such as nitric oxide, superoxide, or arachidonic acid. We chose to test the hypothesis that the intercellular messenger was a reactive oxygen or nitrogen species for several reasons. First, reactive nitrogen species are generated as intercellular messengers during increased firing of neurons in hippocampal long-term potentiation (Chetkovich et al., 1993 ; Schuman and Madison, 1994). Second, MAPK activation is regulated by reactive oxygen and nitrogen species in hippocampal slices (Kanterewicz et al., 1998) and in vitro (Fialkow et al., 1994 ; Stevenson et al., 1994). Third, we have previously found that a reactive oxygen or nitrogen species is responsible for regulation of MBP phosphorylation via protein kinase C during periods of increased neuronal activity in the hippocampus (C.M.A. and J.D.S., unpublished data). To test this hypothesis, we applied reactive oxygen species scavengers and a reactive nitrogen species inhibitor to hippocampal slices, delivered HFS to the alveus, and then assayed the CA1 subregion for changes in MBP phosphorylation at Thr95 (Fig. 5A). We observed a block of the decreased MBP phosphorylation with application of a superoxide scavenger [superoxide dismutase (SOD)], in conjunction with a hydrogen peroxide scavenger (catalase) and a nitric oxide synthase inhibitor N-nitro-L-arginine (L-NOArg) (Fig. 5B ; compare Fig. 1B). These experiments indicate that superoxide, hydrogen peroxide, nitric oxide, or all three together may be the intercellular messengers between neurons and oligodendrocytes that regulates MBP phosphorylation.

To determine if each of these reactive oxygen or nitrogen species contributes to the neuron-glia signaling pathway that leads to the regulation of MBP phosphorylation levels, we applied these scavengers and inhibitor separately to hippocampal slices during HFS of the alveus and measured MBP phosphorylation levels at Thr95 (Fig. 6). We observed that SOD, catalase, and L-NOArg each blocked the decrease in MBP phosphorylation (compare Fig. 1B). The corresponding drug controls—boiled SOD, boiled catalase, or N-nitro-D-arginine (D-NOArg) had no effect on the change in MBP phosphorylation. Similarly, a superoxide spin trap, 5,5-dimethylpyrroline 1-oxide (10 mM), also blocked the changes in MBP phosphorylation (phospho-Thr95, 103 ± 20%, n = 6 ; MBP amount, 110 ± 16%, n = 6), as did the nitric oxide synthase inhibitor N-monomethyl-L-arginine (L-NMMA) but not its inactive enantiomer, N-monomethyl-D-arginine (D-NMMA) (Fig. 6 ; compare Fig. 1B).

image

Figure 6. During high-frequency action potential firing of CA1 axons, superoxide, hydrogen peroxide, and nitric oxide each can contribute to the signaling pathway between CA1 neurons and oligodendrocytes in the alveus. Application of each scavenger or inhibitor alone (solid columns) blocked the decrease in MBP phosphorylation (SOD, 120 units/ml, n = 6 ; catalase, 260 units/ml, n = 6 ; L-NOArg, 50 μM, n = 7 ; L-NMMA, 30 μM, n = 4). However, application of the inactive forms of these scavengers and inhibitors (hatched columns) did not block the decrease in MBP phosphorylation after tetanization of the alveus (boiled SOD, 120 units/ml, n = 6, p < 0.05 by ANOVA ; boiled catalase, 260 units/ml, n = 6, p < 0.05 by ANOVA ; D-NOArg, 50 μM, n = 5, p < 0.05 by ANOVA ; D-NMMA, 30 μM, n = 6, p < 0.05 by ANOVA). MBP amounts were slightly increased in slices adminstered boiled catalase or D-NMMA (boiled catalase, 120 ± 6%, n = 6, p 0.01 by Student’s t test ; D-NMMA, 117 ± 6%, n = 6, p < 0.05 by Student’s t test) but not with any other drug treatment (SOD, 114 ± 19%, n = 6 ; boiled SOD, 106 ± 6%, n = 6 ; catalase, 94 ± 9%, n = 6 ; L-NOArg, 112 ± 11%, n = 9 D-NOArg, 117 ± 9%, n = 5 ; L-NMMA, 100 ± 9%, n = 4).

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In additional in vitro experiments, we tested whether reactive oxygen or nitrogen species donors could have direct effects on MBP immunoreactivity. Application of 10 mM hydrogen peroxide, 1mM 3-morpholinosydnonimine, 1 mMS-nitroso-N-acetylpenicillamine, 1 mM sodium nitroprusside, or 25 μg/ml xanthine oxidase with 100 μ/ml xanthine to purified MBP for 10 min at 32°C had no significant effects on phospho-Thr95 immunoreactivity or total MBP immunoreactivity (data not shown). Thus, this decrease in phospho-MBP immunoreactivity is not due to direct redox effects on the protein. Furthermore, these experiments indicate that each of these reactive oxygen and nitrogen species—superoxide, hydrogen peroxide, and nitric oxide—contributes to the signal from neurons to oligodendrocytes regulating MBP phosphorylation levels during periods of increased neuronal activity.

Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

What is responsible for the generation of the reactive oxygen and nitrogen species in CA1 axons during periods of increased action potential firing ? Our findings implicate nitric oxide synthase, which generates nitric oxide and superoxide in a calcium/calmodulin-dependent manner ; moreover, superoxide can convert to hydrogen peroxide (Pou et al., 1992 ; Xia et al., 1996). As nitric oxide synthase has been found in axons (Dinerman et al., 1994 ; Wendland et al., 1994 ; De Vente et al., 1998), we propose that high-frequency action potential propagation in CA1 axons leads to incrased axonal calcium, which can then activate nitric oxide synthase. To test this model, we examined whether extracellular calcium was necessary for neuron—oligodendrocyte signaling by removing extracellular calcium from the physiologic saline. This manipulation blocked the change in MBP phosphorylation after HFS of the alveus (96 ± 32%, n = 7 ; compare Fig. 1B), with not change in MBP amounts (93 ± 18%, n = 7). This finding suggests that extracellular calcium is necessary for the regulation of MBP phosphorylation during high-frequency action potential firing.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

In these studies, we report that MBP phosphorylation is regulated in the adult hippocampus by periods of increased action potential firing. The phosphorylation of MBP at Thr95, the predominant phosphorylation site by MAPK, decreased in response to HFS but not LFS of the alveus. This regulation of MBP phosphorylation requires action potential generation in axons and is mediated by reactive oxygen species.

Our finding that MBP phosphorylation at Thr95 is regulated during increased neuronal activity is intriguing in light of recent studies of this phosphorylation site. Recently, the three-dimensional structure of MBP has been reconstructed using single-particle electron microscopy (Beniac et al., 1997 ; Ridsdale et al., 1997). Analysis of the structure of MBP has revealed that the triproline domain immediately adjacent to Thr95 is exposed on the outside curve of the molecule and connects two parallel β-strands. This suggests that modifications of this region could have significant effects on the entire protein. Moreover, immunoreactivity against phospho-Thr95 is decreased in multiple sclerosis, suggesting that dephosphorylation at this site could contribute to the destabilization of the myelin sheath (Yon et al., 1996). The locus of the changes in MBP phosphorylation are unknown at present and could be in the myelin (which is likely given the high amounts of MBP present here) or in the cell body. It is tempting to speculate that decreased phosphorylation at Thr95 during periods of high-frequency action potential firing in the hippocampus may decrease compaction of the myelin sheath, which could contribute to the information processing of this circuitry.

The finding that the change in MBP phosphorylation was mediated by reactive oxygen and nitrogen species was surprising, yet indicative of the changing views in the literature regarding these diffusible molecules. Previous studies of the interactions of oligodendrocytes, MBP, and reactive oxygen and nitrogen species have typically reported that application of reactive oxygen or nitrogen species results in cellular death (Kim and Kim, 1991 ; Mitrovic et al., 1994) and inhibition of MBP expression (Mackenzie-Graham et al., 1994). Furthermore, reactive nitrogen species have been found to block action potential conduction along axons (Redford et al., 1997). However, numerous studies have revealed that reactive oxygen and nitrogen species can also serve as signaling molecules in the hippocampus (Böhme et al., 1991 ; O’Dell et al., 1991 ; Haley et al., 1992 ; Chetkovich et al., 1993 ; Schuman and Madison, 1994 ; Klann et al., 1998). Accordingly, we observed no loss in total MBP amounts or decreases in action potential conduction, suggesting that the reactive oxygen and nitrogen species generated during high-frequency firing of CA1 axons are contributing to a physiological signaling pathway between neurons and glial cells rather than promoting pathogenesis.

The regulation of MBP phosphorylation by nitric oxide, superoxide, and hydrogen peroxide suggests that a combination of these species contributes to neuron—oligodendrocyte signaling. The chemistry of these reactive oxygen and nitrogen species is intimately tied together. For example, nitric oxide and superoxide react to form peroxynitrite and can then decompose to a species with hydroxyl radical character (Halliwell and Gutteridge, 1989) ; moreover, superoxide can combine with itself to form hydrogen peroxide (Halliwell and Gutteridge, 1989). Given the intermingling of these three reactive oxygen and nitrogen species, it is difficult to determine definitively the exact reactive oxygen or nitrogen species that resulted in modifications of MBP phosphorylation.

We propose a model in which nitric oxide synthase located in axons is activated by calcium entry during high-frequency action potential firing. This is supported by the observations that nitric oxide synthase is located in axons (Dinerman et al., 1994 ; Wendland et al., 1994 ; De Vente et al., 1998) and that calcium can enter axons through sodium channels or reversal of the sodium/calcium transporter (Waxman and Rithchie, 1993). Nitric oxide synthase activated in the axons could generate the reactive oxygen and nitrogen species that then diffuse to the myelin and result in MAPK inactivation (Pou et al., 1992 ; Xia et al., 1996). Typically, studies have found that MAPK is activated rather than inhibited by reactive oxygen and nitrogen species (Fialkow et al., 1994 ; Stevenson et al., 1994 ; Kanterewicz et al., 1998). However, at high concentrations, reactive oxygen and nitrogen species can have inhibitory effects on protein kinases (Knapp and Klann, 1997). It is possible that MAPK in oligodendrocytes is highly redox-sensitive and that the stimulation paradigm elicited a robust increase in reactive oxygen and nitrogen species, resulting in oxidative inhibition of MAPK or an upstream activator of MAPK. Alternatively, we speculate that reactive oxygen and nitrogen species could have activated an inhibitory component of the MAPK cascade. One potential target could be MAPK inhibitory phosphatase (MKP-1), a MAPK-selective phosphatase whose mRNA levels are increased by application of reactive oxygen species in various systems (Baas and Berk, 1995 ; Guyton et al., 1996 ; Mendelson et al., 1996). Given the delayed change in MBP phosphorylation by MAPK in this paradigm, i.e., 45 min posttetanus, it is possible, albeit highly speculative, that MKP-1 levels could have been increased to subsequently inhibit MAPK, thus decreasing MBP phosphorylation at Thr95. Alternatively, glycogen synthase kinase-3, the one other protein kinase that has been found to phosphorylate Thr95 of MBP (Yu and Yang, 1994), could have been down-regulated to decrease the phosphorylation state of MBP. However, to our knowledge, there are no reports of this protein kinase beeing regulated by reactive oxygen or nitrogen species. Further experiments remain to identify redox-sensitive step in this cascade during neuron—glia signaling.

Since the first description of neuron—glia signaling in the 1970s (Villegas, 1972), we have begun to realize that glial cells are not just passive during information processing in the nervous system, but rather are dynamic players. In these studies, we have found that during periods of increased neuronal activity in the hippocampus, a signaling cascade is initiated in neurons that leads to the regulation of the phosphorylation state of MBP, a key structural protein in myelin. This discovery expands our appreciation of the complex intercellular communication pathways that are activated in the hippocampus during neuronal activity, as well as the potentially unique contribution of oligodendrocytes to information signaling in the hippocampus.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

We thank J. P. Adams, A. E. Anderson, K. Dineley, C. M. Kondratick, B. Mirnikjoo, and J. C. Selcher for critical reading of this manuscript. This work was supported by grant MH 57014 from the National Institutes of Health (to J.D.S.) and a Williams Stamps Farish graduate fellowship (to C.M.A.).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Phosphorylation of MBP Thr95 is regulated by neuronal activity
  6. Time course of the change in MBP phosphorylation
  7. Action potentials are necessary to regulate MBP phosphorylation
  8. All MBP isoforms are regulated by phosphorylation during neuronal activity
  9. Extracellular calcium is necessary for neuron—glia signaling to regulate MBP phosphorylation
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES
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