Chronic Ethanol Tolerance

Hyperexcitability of the CNS is a characteristic component of ethanol withdrawal, and there is good evidence for both a reduction in GABA-mediated inhibitory neurotransmission and an increase in glu-tamate-mediated excitatory neurotransmission following chronic ethanol exposure. Studies using primary neuronal cultures have shown that prolonged exposure to ethanol leads to a supersensitization of NMDA receptor-mediated events, such as Ca2+ influx (49) and Ca2+-dependent processes, including glutamate excitotoxicity (50), and glutamate-NMDA receptor-stimulated nitric oxide (NO) formation (51). Similarly, studies with isolated brain preparations have reported that ethanol-mediated enhancement of GABAA receptor-coupled Cl- flux is decreased following chronic ethanol exposure (52). Thus, chronic ethanol-induced NMDA supersensitivity and GABAA receptor desensitization occur with chronic ethanol exposure and likely contribute to the hyperexcitability of the alcohol-withdrawal syndrome and alcohol neurotoxicity.

There are likely to be multiple mechanisms of chronic ethanol-induced changes in glutamate-NMDA receptor sensitivity. Several studies have reported increased NMDA receptor binding and/or subunit protein levels following chronic ethanol exposure both in vitro and in vivo (53-55), whereas other studies have failed to find any such changes (51,56-58). It has been shown that exposure of cultured cortical neurons to ethanol leads to enhancement of NMDA-stimulated NO formation (but not that stimulated by kainate, AMPA or ionomycin) without a change in receptor density, suggesting that the main factor in chronic ethanol-induced NMDA supersensitivity is related to posttranslational modification of NMDA receptors (51). Similarly, in cells stably transfected with GABAA receptor subunits, ethanol was shown to cause changes in GABAA receptor function similar to those observed in vivo, but no change in surface receptor density (59). This finding is consistent with previous observations in rats (60) and mice (61) chronically exposed to ethanol. Because the transfected cells contain defined GABAA receptor subunits with expression controlled by the dexam-ethasone-sensitive promoter, it is unlikely that ethanol affected subunit expression or produced subunit substitution. Thus, it is possible that posttranslational modification(s) underlie these functional changes. No change in GABAA receptor density following chronic ethanol exposure has been observed in the majority of studies. However, changes in GABAA receptor subunit expression in the brain have been reported, suggesting that subunit changes could play an important role in vivo (62).

Phosphorylation is important in direct and indirect modulation of NMDA receptors and might play a role in synaptic modifications underlying ethanol tolerance. Tyrosine phosphorylation within the C-terminal region of NR2 subunits enhances NMDA currents (63-66) and tyrosine phosphatases reduce NMDA currents (67-69) It is thought that tyrosine kinases and phosphatases participate in a dynamic process that regulates channel activity. Phosphorylation of tyrosine residues within the C-terminal region of the NR2 subunit appears to potentiate NMDA currents by reducing tonic inhibition of the receptor by zinc (70). Calmodulin kinase II has also been reported to phosphorylate the C-terminal region of NR2 (71), but it is not known how this affects channel function. Phosphorylation of the Cl-cassette by protein kinase A (PKA) can enhance NMDA receptor function. In the absence of synaptic activity, it appears that NMDA receptors are phosphorylated by basally active PKA, thus enhancing the activity of quiescent receptors (72,73). Ca2+ influx during receptor activation leads to calcineurin-mediated dephosphorylation and receptor downregulation, which can be overcome by P-adrenoceptor-mediated stimulation of PKA activity (72). As noted above, protein kinase C (PKC) phosphorylation of the Cl-cassette can also regulate channel function. Thus, phosphorylation by a variety of kinases can regulate NMDA receptor sensitivity in ways consistent with ethanol tolerance.

Whether the phosphorylation state of NMDA and GABAa receptors is altered by chronic ethanol exposure is not yet known. Chronic ethanol can increase both PKC levels and activity (74-78) as well as induce heterologous desensitization of cAMP signaling with decreased PKA activity (79-81). Some of these wide-ranging effects of ethanol exposure could relate to changes in subcellular translocation and localization. Ethanol has been shown to stimulate translocation to the nucleus of the catalytic subunit of PKA, where it remains sequestered for as long as ethanol is present (82), and to stimulate translocation of PKC-8 and PKC-e to new intracellular sites (78). Translocation of PKC and PKA isozymes to subcellular anchoring proteins is thought to be important in targeting specific signaling events. Furthermore, PKA and calcineurin (protein phosphatase 2B) are concentrated in postsynaptic densities via a common A-kinase anchoring protein (AKAP79), putting them in position to regulate phosphorylation and/or dephosphorylation of key postsynaptic proteins (83). Clearly, changes in PKA and/or PKC activity and subcellular targeting could play an important role in ethanol-induced changes in synaptic function, including modulation of NMDA and GABAA receptors.

It is estimated that approximately 90% of excitatory glutamatergic synapses in the mammalian brain occur on dendritic spines. Dendritic spines, proposed to be the primary sites of synaptic plasticity in the brain, contain a pronounced postsynaptic density (PSD) enriched in neurotransmitter receptors and associated signal-transducing proteins. Recent studies have demonstrated the importance of cytoskeletal elements and scaffolding proteins in anchoring the molecular components within the PSD. Changes in dendritic spine shape have been correlated with behavioral alterations, such as learning and memory, and may provide a structural basis for plasticity in the brain. Shape changes can occur within seconds and are coupled to changes in synaptic activity (84). In particular, AMPA and NMDA receptor activity is associated with spine dynamics (85). Ethanol may disrupt structural plasticity through alterations in AMPA/NMDA receptor function via direct action on the channels themselves or indirectly through changes in synaptic activity (e.g., enhanced GABAergic and decreased glutamatergic activity). Signaling through Rho GTPases, which regulate the organization of the actin cytoskeleton, appear to play a key role in regulation of actin-based plasticity of dendritic spines (86,87). (Fig. 2). Thus, ethanol-induced changes in NMDA responses could be related to changes in dendritic localization.

Another potentially important process in NMDA and GABAA receptor adaptation during ethanol exposure is receptor-cytoskeletal interaction (88). NMDA receptor NR2 subunits bind to the PDZ domains of a family of closely related postsynaptic density proteins (PSDs) [ PSD-95/synapse-associ-ated protein (SAP)-90, Chapsyn-110/PSD-93, and SAP-102] (89). These proteins appear to function by transporting receptor proteins to the synapse as well as anchoring them at synaptic sites. PSD-95 proteins can undergo head-to-head disulfide linkage resulting in a multimodular scaffold for clustering receptors and/or ion channels and coupling receptor-enzyme complexes and receptor-downstream signaling molecules (90). NMDA receptors are required for activity-dependent synaptic remodeling during development, and studies in hippocampal cultures have shown that the subcellular distribution of NMDA receptors is modulated by receptor activity. Chronic treatment with an NMDA receptor antagonist leads to increased NMDA receptor clustering at synaptic sites. Conversely, spontaneous activity leads to decreased synaptic NMDA receptor clustering (91). Because studies in primary neuronal cell cultures might more closely model developmental processes, an important question to be addressed is whether this activity-dependent redistribution of NMDA receptors also occurs in mature neurons. Whether the functional property of the NMDA receptor itself is altered by clustering and redistribution (i.e., synaptic versus nonsynaptic) is unknown. Receptor redistribution could represent a

Fig. 2. A schematic diagram of glutamate-induced signal transduction associated with brain damage. It is known that excessive glutamate stimulation of NMDA receptors can lead to neuronal death in a form known as excitotoxicity. Rapid death can occur through osmotic damage. Delayed neuronal death is associated with excessive NMDA activation, neuronal depolarization, and other glutamate receptor activation, leading to excessively high levels of calcium that can activate a series of cascades, including oxidative stress and other mechanisms yet to be revealed, that lead to neuronal death. In addition, less dramatic activation of NMDA receptors and/or other factors activate a trophic pathway involving tyrosine kinases including the extracellular-signal regulated kinase-mitogen-activated protein kinase (ERK-MAPKinase) pathway that leads to activation of neuronal survival genes. Thus, alcohol—by changing NMDA receptor sensitivity—can alter the ratios between cell-death pathways activated through glutamate NMDA receptors and cell-survival pathways activated through glutamate NMDA receptors.

Fig. 2. A schematic diagram of glutamate-induced signal transduction associated with brain damage. It is known that excessive glutamate stimulation of NMDA receptors can lead to neuronal death in a form known as excitotoxicity. Rapid death can occur through osmotic damage. Delayed neuronal death is associated with excessive NMDA activation, neuronal depolarization, and other glutamate receptor activation, leading to excessively high levels of calcium that can activate a series of cascades, including oxidative stress and other mechanisms yet to be revealed, that lead to neuronal death. In addition, less dramatic activation of NMDA receptors and/or other factors activate a trophic pathway involving tyrosine kinases including the extracellular-signal regulated kinase-mitogen-activated protein kinase (ERK-MAPKinase) pathway that leads to activation of neuronal survival genes. Thus, alcohol—by changing NMDA receptor sensitivity—can alter the ratios between cell-death pathways activated through glutamate NMDA receptors and cell-survival pathways activated through glutamate NMDA receptors.

novel form of activity-dependent synaptic modification (plasticity), and prolonged inhibition of the NMDA receptor during chronic ethanol exposure might also lead to an increase in NMDA receptor clustering at synaptic sites. This is an intriguing hypothesis that needs testing.

The mitogen-activated protein kinase (MAPK) signaling cascade is one of the most highly conserved signal-transduction systems in eukaryotes. MAPK comprises three signaling modules that include the extracllular-signal regulated kinases (ERKs), stress-activated protein (SAP) kinase/jun N-terminal kinase (JNK), and the p38 kinase pathway. These signaling pathways utilize a small G-protein that couples to activation of a downstream signaling cassette of sequentially acting kinases (92). Ras proteins belong to the superfamily of small GTPases that cycle between inactive GDP-bound states and active GTP-bound states and represent a point of convergence for the transduction and integration of many extracellular signals (93). Ras activity is regulated through activation of guanine nucleotide exchange. Ras-GTP activates the serine/threonine kinase Raf-1 by a complex and poorly understood mechanism that involves its recruitment to the membrane. In addition, Raf-1 has multiple phosphorylation sites that can promote both activation and inhibition. Raf-1 activation initiates a kinase cascade that involves activation of the dual-specificity kinase MAPK/ERK (MEK) that, in turn, activates ERK1 and ERK2 (ERK1/2). Activated ERKs phosphorylate cellular substrates and/or translocate to the nucleus where they regulate transcription of genes critical in proliferation, differentiation, and survival in non-neuronal cells. In neurons, increases in intracellular calcium can activate Ras-ERK signaling, and calcium-dependent NMDA receptor-mediated ERK activation appears to play an important role in NMDA-associated synaptic plasticity and survival (94). Chronic ethanol treatment alters both NMDA receptor sensitivity and activation of ERK cascades through phosphoryla-tion of ERK (Fig. 3). In other examples, NMDA-dependent hippocampal long-term potentiation (LTP) is associated with activation of ERK and is blocked by compounds that inhibit the ability of MEK to activate ERK (95). ERK activation has also been shown to be required for hippocampal dependent-associative learning, and Ras-GRF knockout mice display impaired amygdala-dependent memory consolidation (96,97). Ras-ERK signaling is also associated with regulation of dendrite outgrowth and refinement of neuronal processes. Thus, ERK and other tyrosine kinases can be activated by NMDA receptor-stimulated calcium flux.

Other signaling molecules that have gained attention of late are the lipid kinase phosphoinositol 3 kinase (PI3K), and its downstream target, protein kinase B (PKB). The PI3K pathway consists of a heterodimer of a regulatory subunit (P85) and a catalytic subunit (p110). Activation of PI3K protects cells from apoptosis and is thus considered a survival signal (98). Although the lipid products produced by PI3K can stimulate multiple kinases, its antiapoptotic signal is thought to be mediated through activation of PKB. A number of substrates have been identified for PKB, including the apop-totic protein Bad, the forkhead transcription factor FKHRL1 (a transcriptional regulator of many proapoptotic proteins), glycogen synthase kinase-3 (Gsk3), caspase-9, CREB, IKB-kinase-a (IKKa), and nitric oxide synthase (NOS). Phosphorylation by PKB is often associated with negative regulation by promotion of binding to the protein 14-3-3. Phosphorylation of Bad and FKHRL1 creates a recognition binding site for 14-3-3, which leads to their sequestration upon binding 14-3-3. However, some PKB-mediated inactivations do not involve sequestration by 14-3-3 (such as caspase-9 and Gsk3), and still other substrates are not inactivated by PKB (such as CREB). A number of recent studies have shed light on the fact that a great deal of crosstalk exists between ERK and PKB signaling systems. PI3K-PKB activation is frequently observed to be dependent on Ras activation, and Ras may represent a point of convergence for upstream signals that couple to activation of Raf/ERK and PI3K/PKB. In addition, recent studies have shown that PKB can regulate Raf/ERK signaling, apparently through PKB-mediated phosphorylation of Raf-1 on Ser259 (99). Interestingly, recent evidence suggests that PI3K-PKB activation may also be coupled to NMDA receptor activation.

As discussed earlier, NMDA receptors are major targets of ethanol and appear to play a central role in the effects of acute and chronic ethanol on brain function. Acute ethanol exposure inhibits NMDA

Fig. 3. Hippocampal damage and MAPKinase phosphorylation following binge ethanol treatment of rats (see ref. 28 for details). Shown are sections of hippocampus revealing neuronal damage through amino cupric silver staining and phospho-MAPKinase. (e.g., ERK1/2) through immunohistochemistry using phospho-MAPKinase specific antibodies. Phosphorylation of MAPK-ERK1/2 leads to activation of tyrosine-kinase-mediated neuronal survival-plasticity signals. This figure illustrates how chronic ethanol dependence leads to activation of cell-death pathways and activation of phospho-MAPKinase pathways in similar portions of the hippocampus. Shown is a section of hippocampus with dorsal and ventral (temporal hippocampus) areas. The ventral section here (temporal hippocampus) shows significant brain damage as visualized by silver stain in the upper image (see insets with higher magnification). Less significant amino cupric silver staining cell death is seen in the dorsal hippocampal regions. In the lower image, immunohistochemistry from phospho-MAPKinase also shows a gradient of increased phospho-MAPKinase presence in the ventral dentate gyrus, temporal hippocampal dentate gyrus, as opposed to the more dorsal or upper regions of the hippocampus. Thus, with chronic ethanol treatment, cellular death pathways and cellular survival pathways appear to be activated in similar areas consistent with both signals being related to ethanol-induced NMDA supersensitivity.

Fig. 3. Hippocampal damage and MAPKinase phosphorylation following binge ethanol treatment of rats (see ref. 28 for details). Shown are sections of hippocampus revealing neuronal damage through amino cupric silver staining and phospho-MAPKinase. (e.g., ERK1/2) through immunohistochemistry using phospho-MAPKinase specific antibodies. Phosphorylation of MAPK-ERK1/2 leads to activation of tyrosine-kinase-mediated neuronal survival-plasticity signals. This figure illustrates how chronic ethanol dependence leads to activation of cell-death pathways and activation of phospho-MAPKinase pathways in similar portions of the hippocampus. Shown is a section of hippocampus with dorsal and ventral (temporal hippocampus) areas. The ventral section here (temporal hippocampus) shows significant brain damage as visualized by silver stain in the upper image (see insets with higher magnification). Less significant amino cupric silver staining cell death is seen in the dorsal hippocampal regions. In the lower image, immunohistochemistry from phospho-MAPKinase also shows a gradient of increased phospho-MAPKinase presence in the ventral dentate gyrus, temporal hippocampal dentate gyrus, as opposed to the more dorsal or upper regions of the hippocampus. Thus, with chronic ethanol treatment, cellular death pathways and cellular survival pathways appear to be activated in similar areas consistent with both signals being related to ethanol-induced NMDA supersensitivity.

stimulation of ERK and PKB in vitro (100,101), whereas chronic ethanol exposure has been reported to enhance ERK and PKB activation in vitro (100) and ERK in vivo (Fig. 3). In addition, as discussed below, expression of several growth factors and their receptor tyrosine kinases that couple to ERK and PKB activation are altered by prolonged ethanol exposure. Thus, disruption of ERK and PKB signaling by ethanol may play a key role in ethanol-related neuropathology and ethanol-induced alterations in neurocircuitry and neuroplasticity of the developing and adult brain.

5. GLUTAMATE RELEASE AND ETHANOL DEPENDENCE

In addition to the NMDA supersensitivity that occurs during chronic ethanol treatment and likely contributes to the hyperexcitability of ethanol withdrawal, ethanol also alters extracellular glutamate levels. Acutely ethanol tends to lower CNS extracellular glutamate levels consistent with NMDA receptor antagonism by ethanol (102,103). In alcohol-dependent rats, extracellular levels of glutamate return to control levels. However, during withdrawal, there is a threefold increase in the levels of extracellular glutamate that corresponds in time with the progression of the ethanol-withdrawal syndrome (104-106). Thus, it is likely that the hyperexcitability of the ethanol-withdrawal syndrome involves contributions from supersensitive NMDA receptor responses, increases glutamate release, and blunted GABA inhibitory responses.

6. NEUROTROPHIC FACTORS

Neurotrophins are small protein growth factors that have profound influences upon the development, survival, regulation of function, and plasticity of neurons. The neurotrophin family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5. Although the members of the neurotrophin family are 50-55% homologous, the different neurotrophins promote survival of distinct sets of neurons through distinct receptors. For example, sympathetic neurons respond to NGF and NT-3 but not to BDNF (107), whereas dopamine neurons respond to BDNF and NT-3, but not to NGF (108). All members of the neurotrophin family bind to a low-affinity receptor, p75, and each member binds to a high-affinity trk tyrosine kinase receptor. Signal transduction involves both tyrosine kinase signals as well as internalization and transport of the neurotrophin-receptor complex. Because neurotrophins contribute both to the survival of neurons as well as resistance to toxicity, ethanol-induced changes in neurotrophin levels and/or function could contribute to ethanol-induced neurotoxicity.

Much of the present research on chronic ethanol-induced toxicity and the protective effects of trophic factors have focused on the neurons in the hippocampus and septohippocampal pathways, of which the cholinergic and GABAergic neurons have been shown to be susceptible to neuronal loss and atrophy (19,109-113). These neurons are known to require neurotrophins for normal function and survival. Arendt and colleagues found that 28 wk of ethanol liquid diet (20% v/v) decreased choline acetyltransferase and other cholinergic-specific neuronal markers by 60-80%. However, the reduction in the number of neurons expressing the nerve growth factor receptor component p75 was only reduced 20-30% (22). In contrast to reduced neurotrophin receptors, NGF mRNA was significantly increased throughout the brain, with pronounced increases in the hippocampus, where chronic ethanol treatment increased NGF mRNA approximately twofold (114). Increased NGF expression is known to occur in response to traumatic brain injury (115,116). The increased NGF mRNA levels following chronic ethanol treatment may be the result of neuronal damage induced by ethanol exposure. Arendt et al. (114) observed increased NGF mRNA levels up to 4 wk after being removed from the ethanol diet, which is consistent with a long-term alteration in NGF expression in response to ethanol neurotoxicity. Although increased NGF mRNA levels were associated with dendritic remodeling, the expression of choline acetyltransferase remained decreased. Assuming increased NGF translation, this suggests that the increased NGF level is not robust enough to completely restore choline acetyltransferase expression in chronic ethanol-dam-aged rat brain (114). These studies suggest that chronic ethanol-induced damage induced NGF expression. Walker's group (117) reported that 28 wk of chronic ethanol diet causing septohip-pocampal damage does not change immunoreactive NGF levels or NGF mRNA when animals are sacrificed just after ethanol has cleared from the blood. Interestingly, a third study has found that chronic ethanol treatment of rats leads to elevated NGF content in the hippocampus after 2- or 4-wk exposures, but not after 12-wk exposures (118). Nine months of ethanol liquid diet treatment decreased sciatic nerve NGF by 54% but had no effect on NGF content of the iris, superior cervical ganglion, trigeminal ganglion, or submandibular ganglion (119). Taken together, these studies suggest that NGF levels increase under certain conditions in response to tissue injury, but not sufficiently to correct chronic ethanol-induced damage to the septohippocampal pathways.

Although NGF levels were either unchanged or increased following chronic ethanol treatment, chronic ethanol exposure decreased both neuronal survival (-25%) and neurite-outgrowth (-50%) activities of hippocampal extracts relative to controls, assessed using neuronal culture bioassays (120). At least 21 wk of chronic ethanol treatment were required to reduce neurotrophic activity within the hippocampus. After 28 wk of chronic ethanol treatment, both neurotrophic activity levels and morphological changes resulting from ethanol were found in the septal and hippocampal areas (111,112), suggesting that there may be a relationship between damage and loss of trophic activity. Measurement of mRNA for NT-3, bFGF, and BDNF indicated that only BDNF mRNA appeared to be reduced by 21-28 wk of chronic ethanol treatment (121,122). The loss of BDNF during chronic ethanol treatment likely plays a role in chronic ethanol-induced damage to the septohippocampal pathway (121,122).

Growth factors and NMDA receptors are linked in a number of ways. Growth factors have been found to reduce neuronal sensitivity to NMDA excitotoxicity and oxidative radical formation (123). In contrast, sublethal NMDA stimulation has been shown to have a protective effect on survival of cere-bellar granule cells by inducing BDNF (124,125). BDNF, through a tyrosine kinase mechanism, can actually enhance NMDA receptor responses (126). Ethanol-induced blockade of NMDA receptors during chronic administration could contribute to decreased BDNF expression and increased neuro-toxicity (124). BDNF has been shown to increase the survival of both dopamine neurons (127,128) and serotonin neurons (129); therefore, loss of BDNF would have a deleterious effect on these neuronal populations. For example, in neuronal cultures treatment with NMDA for a short period of time actually increased the density of dopamine neurons, consistent with sublethal NMDA stimulation having a trophic effect (130). However, pretreatment of neuronal cultures with ethanol for 2 d followed by ethanol removal, simulating ethanol withdrawal, resulted in an NMDA neurotoxic response to dopaminergic and serotonergic neurons (131). These results are consistent with other studies showing that chronic ethanol can cause supersensitive NMDA excitotoxicity (50,132). The addition of BDNF to the culture protected against ethanol-induced sensitization to NMDA excitotoxicity in dopamine neurons (131). Chronic ethanol exposure has been found to reduce brain levels of BDNF (121,122). Thus, a mechanism of ethanol neurotoxicity includes reductions in BDNF production sensitizing neurons to insults, including NMDA excitotoxicity.

Receptors for the growth factors are also modified by chronic ethanol treatment. Studies have found that chronic ethanol exposure can decrease levels of p75 neurotrophin receptors (22,133). In contrast, chronic ethanol treatment of rats for 28 wk has been found to increase trk B-like protein expression, suggesting an upregulation of BDNF receptor response elements, perhaps as a compensatory reaction to the decrease in BDNF levels (121). Other studies have found ethanol disruption of neuronal calcium that can be reversed by NGF (134). Ethanol has also been reported to disrupt tyrosine kinase signaling of insulin growth factor receptors (135). Thus, the interaction of ethanol with growth factors could play a key role in ethanol neurotoxicity. The study of growth factor action and its role in ethanol-induced brain damage represents an exciting new area of research with tremendous potential to provide new approaches to the treatment of neurodegeneration.

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