Home Investigators Dorit Ron, Ph.D. Detailed Research Summary
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How does alcohol work? - Location, Location, Location
Unlike other drugs of abuse, alcohol does not have a well-defined site of action. Alcohol is a small molecule that can easily diffuse into all cells. For years, alcohol was thought to act non-specifically. This of course is not the case and many studies have shown that ethanol acts in a very specific manner. The question of how alcohol exerts its specific actions is only now starting to unravel. Over the past 15 years it has become clear that regulation and specificity of complex signal transduction cascades are mediated by compartmentalization of signaling proteins, such as kinases, to specific intracellular sites. This is mediated via protein-protein interactions of signaling proteins with scaffolding proteins. Scaffolding proteins can localize kinases and phosphatases near or away from their corresponding substrates, and can assemble multi-signaling protein complexes, allowing tightly orchestrated events to occur. Since scaffolding proteins are so important for the regulation of various signal transduction cascades, we hypothesized that scaffolding proteins could be a focal point for ethanol’s actions and further hypothesized that ethanol exposure acts to alter the localization and thus function of specific scaffolding proteins. We began to address these questions by determining the effects of ethanol on one such scaffolding protein, RACK1. RACK1 was originally cloned and identified as a specific scaffolding protein for the kinase, betaIIPKC (Ron et al., PNAS, 1994), and is highly expressed in the developing and adult brain (Ashique et al., Brain Research, 2006). Over the years it became apparent that RACK1 acts as a true scaffolding protein that binds to various groups of kinases such as Fyn kinase (Yaka et al., PNAS, 2002), phosphatases, small G proteins, transporters, and cytoplasmic tails of receptors, such as the NR2B subunit of NMDA receptor (Yaka et at., PNAS, 2002). Interestingly, we found that ethanol induces the movement of RACK1 to the nucleus in cultured cells and in vivo, in a mechanism that requires the activation of the cAMP/PKA pathway (Ron et al., FASEB J., 2000; He et al., Mol. Pharmacol., 2002). We further found that as a consequence of RACK1 nuclear compartmentalization, the activity of betaIIPKC is inhibited (Ron et al., FASEB J., 2000), and the expression of certain genes is induced (He et al., Mol. Pharmacol., 2002). Importantly, we found that the altered localization of RACK1 in specific brain regions mediates both the adverse and the beneficial actions of ethanol as described below (Yaka et al., J. Neurosci., 2003; McGough et al., J. Neurosci., 2004).

Fyn kinase – Several years ago we unraveled a novel molecular mechanism by which RACK1 regulates the phosphorylation state and activity of the ligand gated ion channel, NMDA, in the hippocampus. We found that RACK1 localizes the non-receptor protein tyrosine kinase (PTK) Fyn in close proximity to its substrate, the NR2B subunit of the NMDA receptor. RACK1, however, inhibits the ability of Fyn to phosphorylate NR2B, and decreases NMDA receptor-mediated currents in CA1 region of hippocampal slices (Yaka et al., PNAS, 2002; Thornton et al., J. Biol. Chem., 2004). We further found that activation of the cAMP/PKA pathway releases RACK1 from the NMDA receptor complex, leading to enhancement of Fyn phosphorylation of NR2B, and to the enhancement of NMDA receptor activity (Yaka et al., J. Biol. Chem., 2003; Thornton et al., JBC, 2004). The NMDA receptors are involved in synaptic plasticity as well as learning and memory, and are important targets for ethanol-mediated phenotypes such as tolerance, withdrawal, craving and relapse. The molecular mechanisms that underlie ethanol’s actions on the NMDA receptors are largely unknown. Since the activation of the cAMP/PKA pathway resulted in the dissociation of RACK1 from the NMDA receptor (Yaka et al., J. Biol. Chem., 2003), and was required for the nuclear localization of RACK1 in the presence of ethanol (Ron et al., J. Biol. Chem., 2000), we hypothesized that exposure of hippocampal neurons to ethanol will result in the dissociation of the Fyn/RACK1/NR2B-NMDAR complex. As predicted, exposure of hippocampal neurons to ethanol ex vivo and in vivo resulted in the dissociation of NR2B/RACK1/Fyn, and increased phosphorylation of the NR2B subunit by Fyn (Yaka et al., J. Neurosci., 2003). Importantly, RACK1 dissociation and Fyn-mediated phosphorylation of the channel subunit had profound effects on the activity of the channel during and after ethanol exposure (Yaka et al., J. Neurosci., 2003). We obtained further evidence to suggest that this molecular mechanism contributes to the development of acute tolerance in mice (Yaka et al., ACER, 2003). Importantly, we found that alcohol exposure of the dorsal striatum (a brain region associated with habit learninig) leads to Fyn-mediated long-term facilitation of the activity of NR2B-containing NMDARs activity. We also generated evidence to suggest that this process contributes to mechanisms underlying alcohol-drinking behavior (Wang et al., J. Neurosci., 2007 Accepted).

H-Ras – Various studies in recent years have shown that tyrosine phosphorylation of the NR2 subunits of the NMDA receptor mediate the forward trafficking of the channel into the synaptic membranes. We therefore hypothesized that ethanol-mediated phosphorylation of NR2B leads to increased membrane insertion of the subunit. To test this possibility, we examined the localization of the NR2B subunits in hippocampal neurons in the presence and absence of ethanol and found no change in the localization of NR2B. Instead, we observed an increase in the internalization of the NR2A subunit (the other major regulatory subunit of the NMDA receptor in the adult brain), in response to ethanol (Suvarna et al., J. Biol. Chem., 2005). We found that the internalization of NR2A in the presence of ethanol is mediated by the clathrin-dependent endocytic pathway (Suvarna et al., J. Biol. Chem., 2005). Next, we set out to determine a mechanism for ethanol’s actions on the NR2A subunit. Previously, we showed that the interaction between Src kinase and the small GTP binding protein H-Ras leads to the inhibition of Src activity and its ability to phosphorylate its substrate the NR2A subunit (Thornton et al., J. Biol. Chem., 2003). This, in turn, leads to the reduction of the membrane insertion of the channel subunit and to the inhibition of long-term potentiation in hippocampal neurons (Thornton et al., J. Biol. Chem., 2003). We postulated that ethanol-mediated internalization of the NR2A subunit could be due to the activation of H-Ras and the inhibition of Src. As predicted, the H-Ras was active in the presence of ethanol, and ethanol-induced endocytosis of NR2A was dependent on active H-Ras (Suvarna et al., J. Biol. Chem., 2005). Importantly, we found that this molecular mechanism accounts, in part, for the inhibitory actions of ethanol on the NMDA receptor, and we showed that ethanol exposure results in a subunit switch of active NMDA receptors in the hippocampus from NR2A-and NR2B-containing receptors to mainly NR2B-containing channels (Suvarna et al., J. Biol. Chem., 2005). Since the subunit composition of the channel determines its distinct pharmacological properties, this finding has important implications to our understanding of how ethanol alters the function of the NMDA receptors in the brain. Furthermore, our results suggest the Fyn kinase and H-Ras may be targets for drug development to modulate some of the adverse effects of ethanol on the NMDA receptor.

Why most social alcohol drinkers do not develop alcoholism?

BDNF and the dopamine D3 receptor - We found that the activation of the cAMP/PKA pathway in hippocampal neurons leads to RACK1 translocation to the nucleus where it positively regulates the expression of the brain-derived neurotrophic factor (BDNF) (Yaka et al., J. Biol. Chem., 2003). Since ethanol exposure leads to RACK1 translocation, we determine whether ethanol treatment of hippocampal neurons leads to increased expression of BDNF. We found that even low concentrations of ethanol (concentrations that are equivalent to one or two drinks in humans) increase the expression of BDNF in primary hippocampal neurons (McGough et al., J. Neurosci., 2004). Importantly, the BDNF levels were increased in vivo in the dorsal striatum of mice that voluntary consume ethanol vs. mice that consumed only water. To determine the functional consequences of the increase in BDNF expression in response to ethanol, we compared the sensitivity of mice expressing approximately 50% of the BDNF gene (BDNF+/-) and wild-type (WT) mice to ethanol. We found that the reduction of the expression of BDNF leads to increased response of mice to ethanol. The BDNF+/- mice showed an increase in ethanol consumption, increased ethanol-induced sensitization, and increase in the rewarding properties of ethanol compared to their WT littermates (McGough et al., J. Neurosci., 2004). We also inhibited BDNF-mediating signaling by inhibiting the BDNF receptor, TrkB, and found that ethanol consumption was increased compared to vehicle treated mice (Jeanblanc et al., J. Neurosci., 2006). Finally, systemic administration of RACK1 (expressed as a Tat fusion protein) increased the expression of BDNF, and reduced ethanol consumption in a BDNF dependent manner (McGough et al., J. Neurosci., 2004). Taken together, our results suggest that nuclear RACK1, and BDNF are part of a homeostatic pathway that reduces or prevents the development of neuroadaptations that underlie the adverse behavioral phenotypes associated with ethanol exposure (McGough et al., J. Neurosci., 2004). Next, we set out to identify a mechanism that mediates BDNF action to reduce voluntary ethanol intake. We found that in the dorsal striatum, ethanol exposure leads to increases in the expression of the dopamine D3 receptor in a RACK1- and BDNF-dependent manner (Jeanblanc et al., J. Neurosci., 2006). Finally, behavioral data confirmed that the dopamine D3 receptor is part of this homeostatic pathway to control ethanol-drinking behaviors (Jeanblanc et al., J. Neurosci., 2006). We believe that our results provide a molecular mechanism that protects the majority of social drinkers and students that “binge drink” during their college years from becoming alcoholics. To our knowledge, this is the first time that such natural protective mechanisms against alcoholism have been documented.

Identification of the molecular mechanisms mediating the desirable actions of the anti-addiction drug Ibogaine.
GDNF - Ibogaine is an alkaloid extracted from a root bark from the African shrub Iboga. Ibogaine has been used for many years in Africa in religious rituals Interestingly, Ibogaine has been reported to be a powerful anti-addiction drug. Human anecdotal reports suggest that it reduces withdrawal symptoms against heroin and alcohol, and also reduces cravings of most, if not all, drugs of abuse and alcohol. Its effects were also found to be long-lasting. Unfortunately, due to severe side effects such as hallucination, it cannot be used as the “wonder drug” to treat addiction. Several years ago, we set out to identify the mechanism for the desirable actions of Ibogaine. We found that Ibogaine administration decreases voluntary ethanol consumption, and significantly reduces alcohol consumption of rats in a relapse model (He et al., J. Neurosci., 2005). We found that Ibogaine administration leads to the reduction of ethanol drinking behaviors by increasing the expression level of the glial derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) (He et al., J. Neurosci., 2005). Importantly, we found that microinjection of GDNF directly into the VTA reduces ethanol self-administration (He et al., J. Neurosci., 2005). More recently, we showed that the autoregulation of GDNF expression is the mechanism underlying the long-lasting actions of Ibogaine (He and Ron, FASEB J., 2006). These findings open the possibility of using GDNF to develop drugs that will mimic the desirable activities of Ibogaine to fight addiction without producing undesirable side effects.