Andrzej Z. Pietrzykowski

Identification of a BK channel auxiliary protein controlling molecular and behavioral tolerance to alcohol.

Tolerance, described as the loss of drug effectiveness over time, is an important component of addiction. The degree of acute behavioral tolerance to alcohol exhibited by a naïve subject can predict the likelihood of alcohol abuse. Thus, the determinants of acute tolerance are important to understand. Calcium- and voltage-gated (BK) potassium channels, consisting of pore forming α and modulatory β subunits, are targets of ethanol (EtOH) action. Here, we examine the role, at the molecular, cellular, and behavioral levels, of the BK β4 subunit in acute tolerance. Single channel recordings in HEK-293 cells show that, in the absence of β4, EtOH potentiation of activity exhibits acute tolerance, which is blocked by coexpressing the β4 subunit. BK channels in acutely isolated medium spiny neurons from WT mice (in which the β4 subunit is well-represented) exhibit little tolerance. In contrast, neuronal BK channels from β4 knockout (KO) mice do display acute tolerance. Brain slice recordings showed tolerance to EtOHs effects on spike patterning in KO but not in WT mice. In addition, β4 KO mice develop rapid tolerance to EtOHs locomotor effects, whereas WT mice do not. Finally, in a restricted access ethanol self-administration assay, β4 KO mice drink more than their WT counterparts. Taken together, these data indicate that the β4 subunit controls ethanol tolerance at the molecular, cellular, and behavioral levels, and could determine individual differences in alcohol abuse and alcoholism, as well as represent a therapeutic target for alcoholism.

Somatic Localization of a Specific Large-Conductance Calcium-Activated Potassium Channel Subtype Controls Compartmentalized Ethanol Sensitivity in the Nucleus Accumbens

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the β4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel β1 and β4 subunit mRNAs are found in the NAcc, although the signal for β1 is significantly weaker. Immunohistochemistry revealed that β1 subunits were found in both soma and dendrites, whereas β4 appeared restricted to the soma. These findings suggest that the β4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of β4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated αβ4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter αβ1 BK channel-mediated currents. Finally, an EtOH concentration (50 mm) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons.

Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic–neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK α-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

Acute alcohol tolerance is intrinsic to the BKCa protein, but is modulated by the lipid environment.

Ethanol tolerance, in which exposure leads to reduced sensitivity, is an important component of alcohol abuse and addiction. The molecular mechanisms underlying this process remain poorly understood. The BKCa channel plays a central role in the behavioral response to ethanol in Caenorhabditis elegans ( Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele, T. R., Bonci, A., Bargmann, C. I., and McIntire, S. L. (2003) Cell 115, 655-666) and Drosophila (Cowmeadow, R. B., Krishnan, H. R., and Atkinson, N. S. (2005) Alcohol. Clin. Exp. Res. 29, 1777-1786) . In neurons, ethanol tolerance in BKCa channels has two components: a reduced number of membrane channels and decreased potentiation of the remaining channels (Pietrzykowski, A. Z., Martin, G. E., Puig, S. I., Knott, T. K., Lemos, J. R., and Treistman, S. N. (2004) J. Neurosci. 24, 8322-8332) . Here, heterologous expression coupled with planar bilayer techniques examines two additional aspects of tolerance in human BKCa channels. 1) Is acute tolerance observed in a single channel protein complex within a lipid environment reduced to only two lipids? 2) Does lipid bilayer composition affect the appearance of acute tolerance? We found that tolerance was observable in BKCa channels in membrane patches pulled from HEK cells and when they are placed into reconstituted 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine membranes. Furthermore, altering bilayer thickness by incorporating the channel into lipid mixtures of 1,2-dioleoyl-3-phosphatidylethanolamine with phosphatidylcholines of increasing chain length, or with sphingomyelin, strongly affected the sensitivity of the channel, as well as the time course of the acute response. Ethanol sensitivity changed from a strong potentiation in thin bilayers to inhibition in thick sphingomyelin/1,2-dioleoyl-3-phosphatidylethanolamine bilayers. Thus, tolerance can be an intrinsic property of the channel protein-lipid complex, and bilayer thickness plays an important role in shaping the pattern of response to ethanol. As a consequence of these findings the protein-lipid complex should be treated as a unit when studying ethanol action.

The Role of microRNAs in Drug Addiction: A Big Lesson from Tiny Molecules

Alcoholism is a multifactorial disease of unclear molecular underpinnings. Currently, we are witnessing a major shift in our understanding of the functional elements of the genome, which could help us to discover novel insights into the nature of alcoholism. In humans, the vast majority of the genome encodes non-protein-coding DNA with unclear function. Recent research has started to unveil this mystery by describing the functional relevance of microRNAs, and examining which genes are regulated by non-protein-coding DNA. Here, I describe alcohol regulation of microRNAs and provide examples of microRNAs that control the expression of alcohol-relevant genes. Emphasis is put on the potential of microRNAs in explaining the polygenic nature of alcoholism and prospects of microRNA research and future directions of this burgeoning field.

Interleukin‐4 activates large‐conductance, calcium‐activated potassium (BKCa) channels in human airway smooth muscle cells

Large‐conductance, calcium‐activated potassium (BKCa) channels are regulated by voltage and near‐membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper−2 (Th2) cytokine, interleukin (IL)‐4, is an important mediator of airway inflammation, we investigated whether IL‐4 rapidly regulated BKCa activity in normal airway smooth muscle cells. On‐cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin‐4 (50 ng ml−1), IL‐13 (50 ng ml−1) or histamine (10 μm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the α and β1 subunits of BKCa were also performed. Both approaches demonstrated that HBSMC membranes contained large‐conductance channels (>200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BKCa channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL‐4 stimulated rapid, large increases in BKCa channel activity (77.2 ± 63.3‐fold increase, P < 0.05, n= 18). This large potentiation depended on the presence of external calcium. In contrast, IL‐13 (50 ng ml−1) had little effect on BKCa channel activity, but inhibited the effect of IL‐4. Thus, HBSMC contain functional BKCa channels whose activity is rapidly potentiated by the cytokine, IL‐4, but not by IL‐13. These findings are consistent with a model in which IL‐4 rapidly increases near‐membrane calcium concentrations to regulate BKCa activity.

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.

Impulsivity and comorbid traits: a multi-step approach for finding putative responsible microRNAs in the amygdala

Malfunction of synaptic plasticity in different brain regions, including the amygdala plays a role in impulse control deficits that are characteristics of several psychiatric disorders, such as ADHD, schizophrenia, depression and addiction. Previously, we discovered a locus for impulsivity (Impu1) containing the neuregulin 3 (Nrg3) gene, of which the level of expression determines levels of inhibitory control. MicroRNAs (miRNAs) are potent regulators of gene expression, and have recently emerged as important factors contributing to the development of psychiatric disorders. However, their role in impulsivity, as well as control of Nrg3 expression or malfunction of the amygdala, is not well established. Here, we used the GeneNetwork database of BXD mice to search for correlated traits with impulsivity using an overrepresentation analysis to filter for biologically meaningful traits. We determined that inhibitory control was significantly correlated with expression of miR-190b, -28a, -340, -219a, and -491 in the amygdala, and that the overrepresented correlated traits showed a specific pattern of coregulation with these miRNAs. A bioinformatics analysis identified that miR-190b, by targeting an Nrg3-related network, could affect synaptic plasticity in the amygdala, targeting bot impulsive and compulsive traits. Moreover, miR-28a, -340, -219a, and possibly -491 could act on synaptic function by determining the balance between neuronal outgrowth and differentiation. We propose that these miRNAs are attractive candidates of regulation of amygdala synaptic plasticity, possibly during development but also in maintaining the impulsive phenotype. These results can help us to better understand mechanisms of synaptic dysregulation in psychiatric disorders.

Immediate-early alcohol-responsive miRNA expression in Drosophila

Abstract At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in the Drosophila model organism. In our analysis, we combine the power of next-generation sequencing with Drosophila genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 known Drosophila miRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.

Coinciding revolutions: how discovery of non-coding DNA and RNA can change our understanding of addiction

The twenty-first century started with the Big Bang—the first ever sequencing of the human genome in 2000/2003 (Lander et al., 2001; Venter, 2003), and since then our scientific Universe started to expand with the speed of light. This year (2012) the Supernova exploded—the Encode project, a series of thirty papers, trashed the “junk DNA” concept. The project indicates that the vast majority (99%) of the human genome, although non-coding, is not “junk” but is biologically active and essential to maintain fundamental processes in a cell (Bernstein et al., 2012). One of the earlier stellar breakthroughs was the discovery of microRNA, a short RNA species crucial for regulation of gene expression (Lee et al., 1993). With discoveries like these, it becomes more and more obvious that our biological Universe is much more complex and dynamic than we have ever imagined. It is very probable that many biological rules and regulations still await discovery. This is particularly important for our understanding of the mechanisms underlying the development of chronic diseases, like addiction. Addiction is a persistent, neurobiological disease affecting primarily the brain’s reward system, which consists of a circuitry of several interconnecting brain regions, including the prefrontal cortex, nucleus accumbens, ventral tegmental area, amygdala, paraventricular nucleus, and others (Koob and Le Moal, 2001). The reward system is one of the most important physiological features of the brain. We use it constantly, unconsciously, to make countless decisions essential to complete simple and complex tasks, by performing risk/benefit ratio analyses. This powerful motivator drives human and animal behavior, allows an individual to thrive and ultimately, for a species to propagate. Some drugs target the reward system and can drastically change the behavior of individuals vulnerable to addiction, which is characterized as the refocusing of all activities on one goal— the continued intake of the drug. The drug’s effect on the CNS is so powerful that the individual will continue to use it despite any associated adverse consequences. Neurobiological models of addiction try to explain how chronic or frequent exposure to addictive drugs hijacks the elements of the brain reward circuitry and causes transition from voluntary drug-taking to habitual and compulsive drug-seeking (Hyman and Malenka, 2001). During this transition, neuronal adaptations occur, which perpetuate and intensify the disease. Escalating drug intake due to tolerance causes incessant transition of the allostatic set-point, which fails to maintain system stability. Usually, drug addiction and alcohol addiction are described separately, but at the biological level both addictions have common core changes in the reward system through similar neuronal adaptations. However, in either type of addiction, many molecular underpinnings are still unknown. Recently, several papers described the involvement of different non-coding RNAs, primarily microRNA, in unraveling novel molecular mechanisms of this debilitating disease. We put together this Special Issue of Frontiers in Genetics to provide our readers with a comprehensive overview of current research on non-coding RNA in both drug and alcohol addiction. We invited leaders in this dynamic new field to write a series of reviews, and other prominent scientists to share their views of this subject through a series of editorials. This special issue is divided into five chapters: alcohol, nicotine, cocaine, morphine, and bioinformatics. Alcohol is a simple product of yeast fermentation, and is fairly easy to obtain. People have been using alcohol probably from the beginning of recorded history. Alcohol addiction (alcoholism) is the oldest and most widespread type of addiction. In this special issue, Nunez and Mayfield (2012) describe microRNA signatures in the brains of alcoholics and the process of microRNA modulation of epigenetic nuclear mechanisms. Miranda (2012) discusses teratological consequences of microRNA disregulation by alcohol during fetal development. The editorial by Reilly (2012) explains how studying microRNA can provide new perspectives and treatments of alcoholism and Fetal Alcohol Spectrum Disorder (FASD). Nicotine is primarily inhaled during the smoking of dried tobacco leaves (Nicotiana tabacum), but also can be consumed in other forms. Nicotine addiction has spread quickly around the world in the post-Columbian era, becoming a major, presentday health issue in many countries. According to Maccani and Knopik (2012), detrimental effects of nicotine start very early— in utero. They describe in their review how maternal cigarette smoking during pregnancy alters expression of selected microRNAs in placenta. They also discuss the effect of cigarette smoking onmicroRNA and long non-coding RNA (lncRNA) expression in the epithelium of airways, which is important in the pathogenesis of cancer. Their review indicates that environmentally regulated epigenetic changes affect health throughout the course of one’s life, while an editorial by Ehringer (2012) describes the presence of “critical periods” during which the environmental effect is particularly strong. Morphine is the most abundant alkaloid found in opium (opiate), a product of the poppy fruit (Papaver somniferum). Morphine has been used for centuries to treat pain. However, morphine and related products have very potent addictive properties. Currently, addiction to morphine and related drugs is rising, mainly in affluent countries. Several microRNAs have been shown to be important in morphine actions. In this Special Issue,