Clinical Neurochemistry, Department of Psychiatry and Psychotherapy, University of Würzburg, Füchsleinstrasse 15, 97080 Würzburg, Germany,
1 Department of Neuropsychiatry, Sapporo Medical University, S.1, W. 16, Chuo-ku, Sapporo, 060-8543 Japan and
2 State Hospital of Psychiatry and Neurology, Mauer, A-3362 Mauer, Austria
Received 28 March 2000; in revised form 19 June 2000; accepted 8 July 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increased adenylyl cyclase (AC) activity via activation of stimulatory G protein (Gs) is induced by acute exposure to ethanol, whereas chronic exposure to ethanol attenuates AC activity through inactivation of Gs in cells and tissues (Saito et al., 1987; Chung et al., 1989
; Ravin, 1993
). Previous studies with post-mortem human brains reported decreased amount and functions of the G proteins in the cerebral cortex of alcoholics (Ozawa et al., 1993
, 1994
). Moreover, a reduced level of the type I adenylyl cyclase (AC-I) has been found in the temporal cortex of alcoholics (Hashimoto et al., 1998
). These findings suggest a disturbance of the cAMP signal transduction mediated by the dysfunctional G proteins and the impaired particular AC isoform in alcoholic brain.
Regarding the effect of ethanol exposure on downstream events of cAMP signalling, it was reported that chronic treatment with ethanol resulted in an attenuation of the phosphorylation of CREB and its CRE binding activity in rat brain (Yang et al., 1996, 1998a
,b
). These observations suggest the possibility that the impaired cAMP signalling, including reduced CREB-dependent gene transcription, may be involved in the pathophysiology of alcoholism. Nonetheless, very few studies have directly investigated post-second messenger signalling pathways in alcoholic brain. In the present study, we examined the amounts of total CREB and the phosphorylated form of this protein by immunoblotting in homogenate preparations from post-mortem frontal and temporal cortices, and cerebella obtained from alcoholics and age-matched controls. The same brain preparations were also analysed with antibodies to neurofilament 200 (NF-200) and glial fibrillary acidic protein (GFAP) in order to examine the selectivity of results with respect to the general level of neuronal or glial protein expression respectively.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Gel electrophoresis and immunoblotting
The proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and electrophoretically transferred to nitrocellulose membranes for subsequent immunoblotting as previously described. In brief, cortical homogenates were immunoblotted for CREB (30 µg used) (Dowlatshahi et al., 1999; Yamamoto-Sasaki et al., 1999
), for GFAP and NF-200 (30 µg used) (Haug et al., 1996
), respectively. Homogenates were subjected to SDSPAGE with 10% (for CREB proteins and GFAP) or 412% (for NF-200) polyacrylamide gels at 125 V for 2 h. Proteins were transferred to nitrocellulose membranes at 30 V for 75 min at room temperature. The membranes were blocked in 5% bovine serum albumin (BSA)/Tris-buffered saline (TBS) buffer for 1 h at room temperature and then incubated in 3% BSA/TBS buffer overnight at 4°C with either phosphorylated CREB antiserum (1:1000 dilution) or total CREB antiserum (1:2500 dilution) (both Upstate Biotechnology), GFAP antiserum (1:5000 dilution) or NF-200 antiserum (1:5000 dilution) (both from Sigma). Membranes were washed and incubated with secondary antibody, anti-rabbit Ig HRP-linked F(ab')2 (Amersham) diluted to either 1:1000 for CREB or 1:10 000 for NF-200 and GFAP in 3% BSA/TBS buffer for 1 h at room temperature. Immunoreactive bands were detected with the enhanced chemiluminescence (ECL) system (Amersham) and analysed by laser densitometry (NIH 1.55 image analysis system). Linearity of our immunoblots was tested using control human cortical homogenate. In the immunoblots using the standard homogenate, the immunoreactivities showed linearity within the ranges 575 µg for CREB proteins and 1050 µg for GFAP and NF-200.
Statistical analyses
Results are given as the means ± SEM. The data were analysed by Welch's t-test and values of P < 0.05 were taken to indicate statistical significance of differences between groups. The effects of age and post-mortem interval on each immunoreactivity were determined by Spearman's rank order correlation analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CREB proteins
Figure 1 shows representative immunoblots of CREB proteins, NF-200 and GFAP at either expected molecular weight in frontal cortex from alcoholics and control subjects. Immunoblotting with the anti-CREB and the anti-phosphorylated CREB antibodies yielded two bands at apparent molecular weights of 43 kDa and a non-specified band at 55 kDa (Dowlatshahi et al., 1999
; Yamamoto-Sasaki et al., 1999
). The two inducible bands with relative molecular weight of 43 kDa may be splice variants of CREB, i.e. CREB
and CREB
, which differ in sequence by 14 amino acids (Ginty et al., 1993
). Both immunoreactive bands were included in the quantification by densitometry.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have also measured the immunoreactivities of the NF-200 and GFAP as indicators of general glial and neuronal changes in the samples used. Influence of such a general cell alteration in the brain preparations on the results can be ruled out, since immunoblotting studies failed to show any significant differences in NF-200 and GFAP levels between alcoholics and control subjects in each brain region examined.
Concerning the effect of chronic ethanol treatment on cAMP response element (CRE)DNA binding activity of brain CREB, it was found that chronic ethanol treatment had no effect on the CREDNA binding activity in rat cerebellum (Yang et al., 1996), but it was also observed that chronic ethanol exposure impaired CREDNA binding activity in rat striatum (Yang et al., 1998b
). On the other hand, it was reported that acute or chronic ethanol treatment has no significant effect on CREDNA binding activities in the rat cortex, whereas both CREDNA binding activity and the expression of CREB target, i.e. the protein level of BDNF, are decreased in the same region during ethanol withdrawal with peak reductions at 24 h after cessation of chronic ethanol intake (Pandey et al., 1999
). It was thus a logical consequence to look for possible alterations in expression of the CREB protein and CREB phosphorylation state after chronic ethanol exposure in animals and human subjects.
Effects of chronic ethanol intake on the levels of brain CREB were previously investigated in experimental animals. It was observed that there were no significant changes in the levels of total or phosphorylated CREB, but induction of CREB phosphorylation by an acute ethanol challenge was markedly attenuated (50%) in the rat striatum after chronic exposure to ethanol (Yang et al., 1998a). It was also reported that chronic ethanol exposure resulted in a 25% decrease in the level of phosphorylated CREB and in a 50% reduction in the induction of CREB phosphorylation by a subsequent acute ethanol challenge in the granule cell layer of the rat (Yang et al., 1998b
). It was also demonstrated that the attenuated CREB phosphorylation was associated with reduction in the levels of the catalytic subunit of protein kinase A (PKA) and calcium/ calmodulin-dependent kinase IV (CaMKIV), which are known to phosphorylate CREB on Ser-133 (Yang et al., 1998b
).
Our present study has found no significant differences in the levels of phosphorylated or total CREB in the frontal and temporal cortices, nor in cerebella of alcoholics relative to control subjects. In the previous study using rat cerebellum, Yang et al. (1998b) also reported that levels of protein phosphatase 1 (PP-1), which dephosphorylates the phosphorylated CREB, did not alter in the nuclear extracts of cerebellum after chronic and acute ethanol exposure. These authors, however, did not use inhibitors of PP-1 in the preparation of nuclear extracts, whereas we did in the preparation of homogenates. This could explain the difference in the level of phosphorylated CREB between these two studies. Another potential reason for the discrepancy between the results in rat and human subjects is that of species differences in the expression ratio of granule cells to Purkinje cells in cerebellum. Furthermore, we have used whole-cell homogenates for immunoblotting to avoid loss of the CREB proteins during preparation, and this may have a bearing on the results obtained. Future studies concerning alterations in levels and activities of PKA and CaMKIV in human alcoholic brain could provide more information on this point.
In most alcoholic patients examined in the present study, the time from cessation of drinking to death was very short, and it is thus possible to consider the subjects as having been in the withdrawal state. The unchanged amounts of CREB proteins observed in alcoholic cortices and cerebella in the present study seem to be inconsistent with the previous finding in rat cortex showing decreased CREDNA binding activity during ethanol withdrawal (Pandey et al., 1999). However, it has been suggested that phosphorylation of CREB at Ser-133 induces a conformational change of the protein from an inactive to an active form that specifically stimulates the transcriptional activity without affecting its DNA binding properties (Gonzalez et al., 1991
). Therefore, the reduction of CREDNA binding activity observed in the rat cortex is unlikely to be caused by the alteration in the levels of CREB proteins, and it is possible that the unaltered level of phosphorylated CREB in our present study may compensate the disturbed adenylyl cyclase activity and levels of the enzymes in alcoholic brain (Saito et al., 1987
; Ravin, 1993
; Hashimoto et al., 1998
).
Both CREB gene and AC-I gene knock-out animals show impaired spatial and long-term memory, indicating that cAMP signalling plays an essential role in the mechanism of neural plasticity (Bourtchuladze et al., 1994; Wu et al., 1995
). Addiction is characterized by the compulsive use of a drug, which is presumably due to certain adaptive changes that arise in neurons of specific brain areas (Nestler and Duman, 1995
; Self and Nestler, 1995
). Our observations show that neural adaptation to chronic ethanol intake in human cerebral cortex is not directly reflected by a change in the level of CREB and its phosphorylation. However, an unchanged protein level does not always imply an intact protein function. It might be possible that the neuroadaptation to chronic ethanol exposure in the cortical regions of alcoholics will become manifest as altered function of the CREB when the conditioned stimulus, i.e. ethanol, is given, as demonstrated in animal brain (Yang et al., 1998a
,b
), and the alterations in brain CREB physiology might change expression of genes that are dependent on CREB for transcription. This proposition should be elucidated by further studies on the functional quality of the CREB in alcoholic brain.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bourtchuladze, R., Frenguelli, B., Blendy, J., Cioffi, D., Schutz, G. and Silva, A. J. (1994) Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. Cell 79, 5968.[ISI][Medline]
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248254.[ISI][Medline]
Chung, C. T., Tamarkin, L., Hoffman P. L. and Tabakoff, B. (1989) Ethanol enhancement of isoproterenol-stimulated melatonin and cyclic AMP release from cultured pineal glands. Journal of Pharmacology and Experimental Therapeutics 249, 1622.[Abstract]
Dowlatshahi, D., MacQueen, G. M., Wang. J.-F., Reiach, J. S. and Young, L. T. (1999) G protein-coupled cyclic AMP signaling in postmortem brain of subjects with mood disorders: Effects of diagnosis, suicide, and treatment at the time of death. Journal of Neurochemistry 73, 11211126.[ISI][Medline]
Ginty, D. D., Kornhauser, J. M., Thompson, M. A., Bading, H., Mayo, K. E., Takahashi, J. S. and Greenberg, M. E. (1993) Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260, 238241.[ISI][Medline]
Gonzalez, G. A., Menzel, P., Leonard, J., Fischer, W. H. and Montminy, M. R. (1991) Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB. Molecular and Cellular Biology 11, 13061312.[ISI][Medline]
Gsell, W., Lange, K. W., Pfeuffer, R., Heckers, H., Heinsen, H., Senitz, D., Jellinger, K., Ransmayr, G., Wichart, I., Vock, R., Beckmann H. and Riederer, P. (1993) How to run a brain bank: A report from the Austro-German brain bank. Journal of Neural Transmission 39 (Suppl.), 3170.
Hashimoto, E., Frölich, L., Ozawa, H., Saito, T., Maurer, K., Böning, J., Takahata, N. and Riederer, P. (1998) Reduced immunoreactivity of type I adenylyl cyclase in the postmortem brains of alcoholics. Alcoholism: Clinical and Experimental Research 22 (Suppl. to No. 3), 88S92S.[ISI][Medline]
Haug, L. S., Østvold, A. C., Cowburn, R. F., Garlind, A., Winblad, B., Bogdanovich, N. and Walaas, S. I. (1996) Decreased inositol (1,4,5)-triphosphate receptor levels in Alzheimer's disease cerebral cortex: Selectivity of changes and possible correlation to pathological severity. Neurodegeneration 5, 169176.[ISI][Medline]
Hoffman, P. L. and Tabakoff, B. (1990) Ethanol and guanine nucleotide binding proteins: a selective interaction. FASEB Journal 4, 26122622.
Koob, G. F., Sanna, P. P. and Bloom, F. E. (1998) Neuroscience of addiction. Neuron 21, 467476.[ISI][Medline]
Nestler, E. J. and Duman, S. (1995) Intracellular messenger pathways as mediators of neural plasticity. In Psychopharmacology: The Fourth Generation of Progress, Bloom, F. E. and Kupfer, D. J. eds, pp. 695704. Raven Press, New York.
Ozawa, H., Katamura, Y., Hatta, S., Saito, T., Katada, T., Gsell, W., Frölich, L., Takahata, N. and Riederer, P. (1993) Alterations of guanine nucleotide-binding proteins in post-mortem human brain in alcoholics. Brain Research 620, 174179.[ISI][Medline]
Ozawa, H., Saito, T., Hatta, S., Hashimoto, E., Frölich, L., Ohshika, H., Takahata, N. and Riederer, P. (1994) Reduced sensitivity to ethanol of Gs and Gi/o
in the cerebral cortex of alcoholic patients. Alcohol and Alcoholism 29 (Suppl.), 9397.[ISI][Medline]
Pandey, S. C., Zhang, D., Mittal, N. and Nayyar, D. (1999) Potential role of the gene transcription factor cyclic AMP-responsive element binding protein in ethanol withdrawal-related anxiety. Journal of Pharmacology and Experimental Therapeutics 288, 866878.
Ravin, R. A. (1993) Ethanol-induced desensitization of adenylyl cyclase: Role of the adenosine receptor and GTP-binding proteins. Journal of Pharmacology and Experimental Therapeutics 264, 977983.[Abstract]
Saito, T., Lee, J. M., Hoffman, P. L. and Tabakoff, B. (1987) Effects of chronic ethanol treatment on the beta-adrenergic receptor-coupled adenylate cyclase system of mouse cerebral cortex. Journal of Neurochemistry 48, 18171822.[ISI][Medline]
Saito, T., Katamura, Y., Ozawa, H., Hatta, S. and Takahata, N. (1994) Platelet GTP-binding protein in long-term abstinent alcoholics with an alcoholic first-degree relative. Biological Psychiatry 36, 495497.[ISI][Medline]
Self, D. W. and Nestler, E. J. (1995) Molecular mechanisms of drug reinforcement and addiction. Annual Reviews in Neuroscience 18, 463495.[ISI][Medline]
Wand, G. S., Diehl, A. M., Levine, M. A., Wolfgang, D. and Sany, S. (1993) Chronic ethanol treatment increases expression of inhibitory G-proteins and reduces adenylyl cyclase activity in the central nervous system of two lines of ethanol-sensitive mice. Journal of Biological Chemistry 268, 25952601.
Wu, Z.-L., Thomas, S. A., Villacres, E. C., Xia, Z., Simmons, M. L., Chavkin, C., Palmiter, R. D. and Storm, D. R. (1995) Altered behavior and long-term potentiation in type I adenylyl cyclase mutant mice. Proceedings of the National Academy of Sciences of the USA 92, 220224.[Abstract]
Yamamoto-Sasaki, M., Ozawa, H., Saito, T., Rösler, M. and Riederer, P. (1999) Impaired phosphorylation of cyclic AMP response element binding protein in the hippocampus of dementia of the Alzheimer type. Brain Research 824, 300303.[ISI][Medline]
Yang, X., Diehl, A. M. and Wand, G. S. (1996) Ethanol exposure alters the phosphorylation of cyclic AMP responsive element binding protein and cyclic AMP responsive element binding activity in rat cerebellum. Journal of Pharmacology and Experimental Therapeutics 278, 338346.[Abstract]
Yang, X., Horn, K., Baraban, J. M. and Wand, G. S. (1998a) Chronic ethanol administration decreases phosphorylation of cyclic AMP response element-binding protein in granule cells of rat cerebellum. Journal of Neurochemistry 70, 224232.[ISI][Medline]
Yang, X., Horn, K. and Wand, G. S. (1998b) Chronic ethanol exposure impairs phosphorylation of CREB and CRE-binding activity in rat striatum. Alcoholism: Clinical and Experimental Research 22, 382390.[ISI][Medline]
Zafra, F., Lindholm, D., Castren, E., Hartikka, J. and Thoenen, H. (1992) Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes. Journal of Neuroscience 12, 47934799.[Abstract]