DlgS97/SAP97, a neuronal isoform of discs large, regulates ethanol tolerance.

From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, we identified intolerant (intol), a novel allele of discs large 1 (dlg1). Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly's brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol's sedative/hypnotic effects. We propose that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity.

An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol.

Anaplastic lymphoma kinase (Alk) is a gene expressed in the nervous system that encodes a receptor tyrosine kinase commonly known for its oncogenic function in various human cancers. We have determined that Alk is associated with altered behavioral responses to ethanol in the fruit fly Drosophila melanogaster, in mice, and in humans. Mutant flies containing transposon insertions in dAlk demonstrate increased resistance to the sedating effect of ethanol. Database analyses revealed that Alk expression levels in the brains of recombinant inbred mice are negatively correlated with ethanol-induced ataxia and ethanol consumption. We therefore tested Alk gene knockout mice and found that they sedate longer in response to high doses of ethanol and consume more ethanol than wild-type mice. Finally, sequencing of human ALK led to the discovery of four polymorphisms associated with a low level of response to ethanol, an intermediate phenotype that is predictive of future alcohol use disorders (AUDs). These results suggest that Alk plays an evolutionary conserved role in ethanol-related behaviors. Moreover, ALK may be a novel candidate gene conferring risk for AUDs as well as a potential target for pharmacological intervention.

Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse.

BACKGROUND: Previous work from our laboratory demonstrated a role for the Drosophila Lim-only (dLmo) gene in regulating behavioral responses to cocaine. Herein, we examined whether dLmo influences the flies' sensitivity to ethanol's sedating effects. We also investigated whether 1 of the mammalian homologs of dLmo, Lmo3, is involved in behavioral responses to ethanol in mice. METHODS: To examine dLmo function in ethanol-induced sedation, mutant flies with reduced or increased dLmo expression were tested using the loss of righting (LOR) assay. To determine whether mouse Lmo3 regulates behavioral responses to ethanol, we generated transgenic mice expressing a short-hairpin RNA targeting Lmo3 for RNA interference-mediated knockdown by lentiviral infection of single cell embryos. Adult founder mice, expressing varying amounts of Lmo3 in the brain, were tested using ethanol loss-of-righting-reflex (LORR) and 2-bottle choice ethanol consumption assays. RESULTS: We found that in flies, reduced dLmo activity increased sensitivity to ethanol-induced sedation, whereas increased expression of dLmo led to increased resistance to ethanol-induced sedation. In mice, reduced levels of Lmo3 were correlated with increased sedation time in the LORR test and decreased ethanol consumption in the 2-bottle choice protocol. CONCLUSIONS: These data describe a novel and conserved role for Lmo genes in flies and mice in behavioral responses to ethanol. These studies also demonstrate the feasibility of rapidly translating findings from invertebrate systems to mammalian models of alcohol abuse by combining RNA interference in transgenic mice and behavioral testing.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

Protein Phosphatase 2a and glycogen synthase kinase 3 signaling modulate prepulse inhibition of the acoustic startle response by altering cortical M-Type potassium channel activity.

There is considerable interest in the regulation of sensorimotor gating, since deficits in this process could play a critical role in the symptoms of schizophrenia and other psychiatric disorders. Sensorimotor gating is often studied in humans and rodents using the prepulse inhibition of the acoustic startle response (PPI) model, in which an acoustic prepulse suppresses behavioral output to a startle-inducing stimulus. However, the molecular and neural mechanisms underlying PPI are poorly understood. Here, we show that a regulatory pathway involving protein phosphatase 2A (PP2A), glycogen synthase kinase 3 beta (GSK3beta), and their downstream target, the M-type potassium channel, regulates PPI. Mice (Mus musculus) carrying a hypomorphic allele of Ppp2r5delta, encoding a regulatory subunit of PP2A, show attenuated PPI. This PPP2R5delta reduction increases the phosphorylation of GSK3beta at serine 9, which inactivates GSK3beta, indicating that PPP2R5delta positively regulates GSK3beta activity in the brain. Consistently, genetic and pharmacological manipulations that reduce GSK3beta function attenuate PPI. The M-type potassium channel subunit, KCNQ2, is a putative GSK3beta substrate. Genetic reduction of Kcnq2 also reduces PPI, as does systemic inhibition of M-channels with linopirdine. Importantly, both the GSK3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)1H-pyrrole-2,5-dione (SB216763) and linopirdine reduce PPI when directly infused into the medial prefrontal cortex (mPFC). Whole-cell electrophysiological recordings of mPFC neurons show that SB216763 and linopirdine have similar effects on firing, and GSK3 inhibition occludes the effects of M-channel inhibition. These data support a previously uncharacterized mechanism by which PP2A/GSK3beta signaling regulates M-type potassium channel activity in the mPFC to modulate sensorimotor gating.

Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.

The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster.

BACKGROUND: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants. METHODS: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure. RESULTS: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects. CONCLUSIONS: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

Rapid and chronic: two distinct forms of ethanol tolerance in Drosophila.

BACKGROUND: Ethanol tolerance, defined as a reduction in the intensity of the effects of ethanol upon continuous or repeated exposure, is a hallmark of alcoholism. Tolerance may develop at the cellular or neural systems levels. The molecular changes underlying ethanol tolerance are not well understood. We therefore explored the utility of Drosophila, with its accessibility to genetic, molecular, and behavioral analyses, as a model organism to study tolerance development in response to different ethanol-exposure regimens. METHODS: We describe a new assay that quantifies recovery from ethanol intoxication in Drosophila. Using this recovery assay, we define ethanol pre-exposure paradigms that lead to the development of tolerance. We also use the inebriometer, an assay that measures the onset of intoxication, to study the effects of pharmacological and genetic manipulations on tolerance development. RESULTS: We show that flies develop different forms of ethanol tolerance: rapid tolerance, induced by a single short exposure to a high concentration of ethanol, and chronic tolerance, elicited by prolonged exposure to a low concentration of the drug. Neither rapid nor chronic tolerance involves changes in ethanol pharmacokinetics, implying that they represent functional rather than dispositional tolerance. Chronic and rapid tolerance can be distinguished mechanistically: chronic tolerance is disrupted by treatment with the protein synthesis inhibitor cycloheximide, whereas rapid tolerance is resistant to this treatment. Furthermore, rapid and chronic tolerance rely on distinct genetic pathways: a mutant defective for octopamine biosynthesis shows reduced rapid tolerance but normal chronic tolerance. CONCLUSIONS: Flies, like mammals, develop tolerance in response to different ethanol-exposure regimens, and this tolerance affects both the onset of and the recovery from acute intoxication. Two forms of tolerance, rapid and chronic, are mechanistically distinct, because they can be dissociated genetically and pharmacologically.