Alfredo Ghezzi

Drug-Induced Epigenetic Changes Produce Drug Tolerance

Tolerance to drugs that affect neural activity is mediated, in part, by adaptive mechanisms that attempt to restore normal neural excitability. Changes in the expression of ion channel genes are thought to play an important role in these neural adaptations. The slo gene encodes the pore-forming subunit of BK-type Ca2+-activated K+ channels, which regulate many aspects of neural activity. Given that induction of slo gene expression plays an important role in the acquisition of tolerance to sedating drugs, we investigated the molecular mechanism of gene induction. Using chromatin immunoprecipitation followed by real-time PCR, we show that a single brief sedation with the anesthetic benzyl alcohol generates a spatiotemporal pattern of histone H4 acetylation across the slo promoter region. Inducing histone acetylation with a histone deacetylase inhibitor yields a similar pattern of changes in histone acetylation, up-regulates slo expression, and phenocopies tolerance in a slo-dependent manner. The cAMP response element binding protein (CREB) is an important transcription factor mediating experience-based neuroadaptations. The slo promoter region contains putative binding sites for the CREB transcription factor. Chromatin immunoprecipitation assays show that benzyl alcohol sedation enhances CREB binding within the slo promoter region. Furthermore, activation of a CREB dominant-negative transgene blocks benzyl alcohol–induced changes in histone acetylation within the slo promoter region, slo induction, and behavioral tolerance caused by benzyl alcohol sedation. These findings provide unique evidence that links molecular epigenetic histone modifications and transcriptional induction of an ion channel gene with a single behavioral event.

Susceptibility to ethanol withdrawal seizures is produced by BK channel gene expression.

Alcohol withdrawal seizures are part of the symptomatology of severe alcohol dependence and are believed to originate from long‐term neural adaptations that counter the central nervous system depressant effects of alcohol. Upon alcohol withdrawal, however, the increased neural excitability that was adaptive in the presence of alcohol becomes counter‐adaptive and produces an imbalanced hyperactive nervous system. For some individuals, the uncovering of this imbalance by alcohol abstention can be sufficient to generate a seizure. Using the Drosophila model organism, we demonstrate a central role for the BK‐type Ca2+‐activated K+ channel gene slo in the production of alcohol withdrawal seizures.

CREB regulation of BK channel gene expression underlies rapid drug tolerance

Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK‐type Ca2+‐activated K+ channels and has been linked to alcohol‐ and organic solvent‐induced behavioral tolerance in mice, Caenorhabditis elegans (C. elegans) and Drosophila. The cyclic AMP response element‐binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here, we show that benzyl alcohol sedation alters expression of both dCREB‐A and dCREB2‐b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB‐responsive reporter gene, we show that benzyl alcohol sedation increases CREB‐mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase‐inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss‐of‐function allele of dCREB2 eliminates drug‐induced upregulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol‐induced phenotypes. These findings suggest that CREB positively regulates the expression of slo‐encoded BK‐type Ca2+‐activated K+ channels and that this gives rise to behavioral tolerance to benzyl alcohol sedation.

BK channels play a counter-adaptive role in drug tolerance and dependence.

Disturbance of neural activity by sedative drugs has been proposed to trigger a homeostatic response that resists unfavorable changes in net cellular excitability, leading to tolerance and dependence. The Drosophila slo gene encodes a BK-type Ca2+-activated K+ channel implicated in functional tolerance to alcohol and volatile anesthetics. We hypothesized that increased expression of BK channels induced by these drugs constitutes the homeostatic adaptation conferring resistance to sedative drugs. In contrast to the dogmatic view that BK channels act as neural depressants, we show that drug-induced slo expression enhances excitability by reducing the neuronal refractory period. Although this neuroadaptation directly counters some effects of anesthetics, it also causes long-lasting enhancement of seizure susceptibility, a common symptom of drug withdrawal. These data provide a possible mechanism for the long-standing counter-adaptive theory for drug tolerance in which homeostatic adaptations triggered by drug exposure to produce drug tolerance become counter-adaptive after drug clearance and result in symptoms of dependence.

Alcohol-Induced Histone Acetylation Reveals a Gene Network Involved in Alcohol Tolerance

Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

Circadian Genes Differentially Affect Tolerance to Ethanol in Drosophila

BACKGROUND There is a strong relationship between circadian rhythms and ethanol (EtOH) responses. EtOH consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Alcohol Clin Exp Res 2005b, 29, 1550). The Drosophila central circadian pacemaker is composed of proteins encoded by the per, tim, cyc, and Clk genes. Using Drosophila mutant analysis, we asked whether these central components of the circadian clock make the equivalent contribution toward EtOH tolerance and whether rhythmicity itself is necessary for tolerance. METHODS We tested flies carrying mutations in core clock genes for the capacity to acquire EtOH tolerance. Tolerance was assayed by comparing the sedation curves of populations during their first and second sedation. Animals that had acquired tolerance sedated more slowly. Movement was also monitored as the flies breathe the EtOH vapor to determine if other facets of the EtOH response were affected by the mutations. Gas chromatography was used to measure internal EtOH concentration. Constant light was used to nongenetically destabilize the PER and TIM proteins. RESULTS A group of circadian mutations, all of which eliminate circadian rhythms, do not disrupt tolerance identically. Mutations in per, tim, and cyc completely block tolerance. However, a mutation in Clk does not interfere with tolerance. Constant light also disrupts the capacity to acquire tolerance. These lines did not differ in EtOH absorption. CONCLUSIONS Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid EtOH tolerance. However, the role of circadian genes in EtOH tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire EtOH tolerance by some circadian mutations is not merely a downstream effect of a nonfunctional circadian clock; instead, these circadian genes play an independent role in EtOH tolerance.

A role for dynamin in triggering ethanol tolerance

BACKGROUND A prevailing hypothesis is that the set of genes that underlie the endophenotypes of alcoholism overlap with those responsible for the addicted state. Functional ethanol tolerance, an endophenotype of alcoholism, is defined as a reduced response to ethanol caused by prior ethanol exposure. The neuronal origins of functional rapid tolerance are thought to be a homeostatic response of the nervous system that counters the effects of the drug. Synaptic proteins that regulate neuronal activity are an important evolutionarily conserved target of ethanol. METHODS We used mutant analysis in Drosophila to identify synaptic proteins that are important for the acquisition of rapid tolerance to sedation with ethanol. Tolerance was assayed by sedating flies with ethanol vapor and comparing the recovery time of flies after their first sedation and their second sedation. Temperature-sensitive paralytic mutants that alter key facets of synaptic neurotransmission, such as the propagation of action potentials, synaptic vesicle fusion, exocytosis, and endocytosis, were tested for the ability to acquire functional tolerance at both the permissive and restrictive temperatures. RESULTS The shibire gene encodes Drosophila Dynamin. We tested 2 temperature-sensitive alleles of the gene. The shi(ts1) allele blocked tolerance at both the permissive and restrictive temperatures, while shi(ts2) blocked only at the restrictive temperature. Using the temperature-sensitive property of shi(ts2) , we showed that Dynamin function is required concomitant with exposure to ethanol. A temperature-sensitive allele of the Syntaxin 1A gene, Syx1A(3-69), also blocked the acquisition of ethanol tolerance. CONCLUSIONS We have shown that shibire and Syntaxin 1A are required for the acquisition of rapid functional tolerance to ethanol. Furthermore, the shibire gene product, Dynamin, appears to be required for an immediate early response to ethanol that triggers a cellular response leading to rapid functional tolerance.

Homeostatic Cont of Neural Activity: A Drosophila Model for Drug Tolerance and Dependence

Drug addiction is a complex condition of compulsive drug use that results in devastating physical and social consequences. Drosophila melanogaster has recently emerged as a valuable genetic model for investigating the mechanisms of addiction. Drug tolerance is a measurable endophenotype of addiction that can be easily generated and detected in animal models. The counteradaptive theory for drug dependence postulates that the homeostatic adaptations that produce drug tolerance become counteradaptive after drug clearance, resulting in symptoms of dependence. In flies, a single sedation with ethanol or with an organic solvent anesthetic (benzyl alcohol) induces functional tolerance, an adaptation of the nervous system that reduces the effect of these neural depressants. Here we review the role of the BK channel gene (slo) and genes that encode other synaptic proteins in the process of producing functional tolerance. These proteins are predicted to be part of an orchestrated response that involves specific interactions across a highly complex synaptic protein network. The response of the slo gene to drug exposure and the consequence of induced slo expression fit nicely the tenets of the counteradaptive theory for drug tolerance and dependence. Induction of slo expression represents an adaptive process that generates tolerance because it enhances neuronal excitability, which counters the sedative effects of the drugs. After drug clearance, however, the increase in slo expression leads to an allostatic withdrawal state that is characterized by an increase in the susceptibility for seizure. Together, these results demonstrate a common origin for development of drug tolerance and withdrawal hyperexcitability in Drosophila.

Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals.

Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.

Alcohol resistance in Drosophila is modulated by the Toll innate immune pathway

A growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol‐related behaviors. Here, we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder. The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll‐like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila‐specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance whereas decreasing the pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.