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RESEARCH COMPONENT 4:
PI3K/AKT/mTOR

Principal Investigator: Dorit Ron, PhD, Center Director

The first round of funding was aimed to elucidate the role of H-Ras in the nucleus accumbens (NAc) in ethanol-mediated biochemical and behavioral neuroadaptations. We found that acute systemic administration of ethanol as well excessive consumption of ethanol results in the activation of H-Ras in the NA cof mice and rats, and that siRNA-mediated knockdown or pharmacologic inhibition of H-Ras in the NAc leads to the attenuation of ethanol-drinking behaviors in rodents (Ben Hamida J of Neuroscience 2012). We further showed that ethanol-mediated activation of H-Ras leads to the activation of the PI3K/AKT Pathway (Neasta Biol. Psychiatry 2011) resulting in the activation of mTORC1 in the NAc (Nesta et al. PNAS 2010). Importantly, pharmacological inhibition of each of these kinases including the FDA-approved mTORC1 inhibitor, Rapamycin, resulted in the inhibition of excessive ethanol consumption ad seeking (Nesta et al. PNAS 2010, Nesta et al. Biol. Psychiatry 2011, Ben Hamida et al. J of Neuroscience 2012, ). Based on these results we hypothesized that signaling pathways downstream of H-Ras in the NAc play a crucial role in molecular mechanism underlying ethanol-drinking behaviors. Here, we plan to test whether specific pathways that stem of PI3K, AKT and mTORC1 are induced in the NAc of mice and rats in response to repeated cycles of excessive ethanol consumption and withdrawal, and whether these contribute to the development and/or maintenance of ethanol-drinking behaviors. H-Ras/PI3K/AKT and mTORC1 inhibitors are actively being developed by the pharmaceutical industry as therapies against cancer autoimmune diseases and as immunosuppressants. Therefore, we believe that results obtained from these experiments could further lead to the identification and rapid development of new medications to treat alcohol abuse disorders and addiction. Furthermore, the PI3K/AKTmTORC1 pathway is a focal point in various signaling cascades to be investigated within the ACGT. We will, therefore, be able to integrate our findings, share knowledge and reagents and easily collaborate these projects.

Publications

Neasta J., Barak S. Ben Hamida S. and Ron D. mTOR Complex 1: A Key Player in Neuroadaptations Induced by Drugs of Abuse. J. Neurochem. 130(2):172-184, 2014.

Ahmadiantehrani S, Warnault V, Legastelois R, Ron D. From signaling pathways to behavior: the light and dark sides of alcohol. Book Chapter in Neurobiology of Alcohol Dependence, NIAAA. 155-171, 2014.

Barak S, Liu F, Ben Hamida S, Yowell QV, Neasta J, Kharazia V, Janak PH, Ron D. Disruption of alcohol-related memories by mTORC1 inhibition prevents relapse. Nature Neurosci 16(8):1111-7, 2013. See also http://www.nature.com/news/blocking-boozy-memories-reduces-risk-of-relapse-1.13252. PMCID: PMC3725202

Ron D and Messing RO. Signaling pathways mediating alcohol effects. Curr Top Behav Neurosci. 13:87-126, 2013.

Ben Hamida S, Neasta J, Lasek AW, Kharazia V, Zou M, Carnicella S, Janak PH, Ron D. The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors. J Neurosci 32:15849-15858, 2012. (Faculty of 1000 Prime Recommendation: http://f1000.com/prime/717962739#recommendations).

Neasta J, Ben Hamida S, Yowell QV, Carnicella S, Ron D. AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors. Biological Psychiatry 70:575-582, 2011.

Neasta J, Ben Hamida S, Yowell Q, Carnicella S, Ron D. A role for mTOR complex 1 signaling in neuroadaptations underlying alcohol-related disorders. Proc Natl Acad Sci USA 107:20093-20098, 2010.

Suvarna N, Borgland S, Auberson YP, Bonci A, Ron D. Acute ethanol exposure alters functional NMDA receptor NR2 subunit ratio via H-Ras. J Biol Chem 280:31450-31459, 2005.

RESEARCH COMPONENT 5:
PKMzeta

Principal Investigator: Robert O. Messing, MD, Professor, University of Texas at Austin
Co-Investigator: Al Burlingame, Professor, Departments of Chemistry and Pharmaceutical Chemistry,UCSF

This project examines the contribution of atypical PKC (aPKC) isoforms in the NAcb on regulation of alcohol intake. A broad literature supports a role for NAcb dopamine receptors and CB1 and mGluR5 receptors in sustaining alcohol intake in rodent. Our in vitro experiments suggest that dopamine receptor enhancement of NAcb firing in vitro requires an atypical PKC isoform as well as CB1 and mGluR5 receptors.

Thus, we have developed shRNA constructs selective for inhibiting either the aPKC PKC zeta or the aPKC PKC iota/lambda. These are packaged into AAV for infection of rat or mouse NAcb. We then determine the effect of these shRNAs on alcohol intake. We also examine whether the same NAcb aPKC isoform that mediates alcohol intake also mediates the dopamine/cannabinoid enhancement of firing in vitro. In addition, we utilize several transgenic mouse lines where inhibitory constructs for different aPKC isoforms are double-floxed. These mice can then be bred with transgenic mouse CRE lines to allow cell type-selective knockdown. We can also inject a CRE-packaged virus locally within the brain for knockdown in a particular region

By identifying the NAcb signaling molecules through which dopamine receptors sustain alcohol intake, we hope to develop novel therapeutic interventions to counter some of the biological effects of dopamine, but without the more negative side effects that existing dopamine receptor antagonists often show in humans.

RESEARCH COMPONENT 6:
Orexin

Principal Investigator: Frederic “Woody” Hopf, PhD, Associate Adjunct Professor, Neurology Department, UCSF
Consultant (OSC): Patricia Janak, PhD, Professor, Department of Neuroscience, Johns Hopkins University

We are interested in how orexin/hypocretin peptides are able to regulate of pathological forms of alcohol intake, and thus would represent a novel pharmacological therapy to treat Alcohol Use Disorders in humans. Orexin/hypocretins are produced by a small subset of neurons in the hypothalamus, which then project to many brain regions and regulate many different physiological functions including feeding, stress, arousal and sleep. Previous work from our group and others has shown that the orexin system enhances alcohol self-administration and mediates the ability of stress to increase alcohol seeking, in addition to promoting the expression of addiction to other abused substances as well as compulsive feeding and obesity. However, much remains unknown about the molecular signaling pathways activated by orexin receptors, or about the specific brain regions where these orexin receptors act to promote pathological alcohol intake. Thus, we examine how alcohol drinking is controlled by orexin within brain regions that are important regulators of addictive behavior, including the ventral tegmental area (VTA), nucleus accumbens (NAc), amygdala and cortical regions. We also examine how orexin receptors activate downstream signaling pathways, especially the mTorC1-related signaling that is a central focus of Component 4 of the ACGT. Thus, our studies will be of broad interest to the addiction field and other components of the ACGT, and may lead to novel pharmacotherapies for alcoholism and other addictions.

PILOT PROJECT PROGRAM

Allison Xu, PhD, Associate Professor, Diabetes Center, UCSF

Pilot Project: Alcohol-regulated neuronal circuitry underlying alcoholic fatty liver diseases

One of the most serious health consequences of alcohol abuse is the development of liver disease, which could lead to liver failure and death. Hepatic steatosis manifests in almost all heavy alcohol drinkers, and it marks the early stage of liver injury, which subsequently progresses to alcoholic hepatitis, fibrosis and cirrhosis. To date, the mechanisms underlying the development of alcoholic fatty liver are still not welldefined. Our laboratory has recently established a brain-to-liver circuit in regulation of hepatic lipid metabolism under physiological conditions such as starvation and high-fat feeding. We show that Agoutirelated protein (AgRP), a neuropeptide exclusively expressed in the mediobasal hypothalamus, is required for the development of hepatic steatosis via mechanisms independent of food intake and body weight. We have also identified that ankyrin repeat and SOCS box-containing 4 (Asb4) is a novel alcohol-regulated gene. In this pilot and feasibility proposal, we will explore the functional roles of Asb4 in alcoholic fatty liver and understand its regulation by alcohol. We will map the anatomical location of alcohol-responsive Asb4 neurons, and to determine whether their alcohol-responsiveness depends on functional AgRP neuropeptide. We will also investigate whether dynamic regulation of Asb4 by alcohol is required for the development of alcoholic fatty liver. This study will allow us to define a new alcohol-regulated neuronal circuitry, and help understand how alcohol exerts its pathological effects by interacting with regulatory pathways that are normally engaged in energy metabolism.

Kevan Shokat, PhD, Professor and Chair, Department of Cellular and Molecular Pharmacology, HHMI investigator, UCSF

Pilot Project: A New Generation of mTOR Inhibitor to treat Neuroadaptations underlying alcohol-related disorders

Elyssa Margolis, PhD, Adjunct Assistant Professor, Neurology Department, UCSF

Pilot Project: Ventral tegmental area extrasynaptic GABAA receptor role in ethanol reward

The reinforcing properties of all drugs of abuse, including ethanol, are thought to depend upon their ability to stimulate dopamine release. However, ethanol acts on a variety of brain regions and proteins in the brain, and our understanding of the mechanisms which lead to elevated ethanol consumption is incomplete. The ventral tegmental area (VTA) contains the dopaminergic cell bodies which are believed to be critical for the motivational aspects of drug taking, and animals will self administer ethanol directly into the VTA. In spite of this, the synaptic mechanism(s) by which ethanol controls the excitability of VTA neurons and produces motivational signals from the VTA are unsettled issues. Here we propose to determine the role that extrasynaptic GABA-A receptors play in ethanol control of VTA neurons and to test if these receptors contribute to the motivational properties of ethanol in the VTA. Because ethanol modulates GABA-A receptors at much lower doses (as low as 3 mM) than the previously reported effects of ethanol on VTA neurons (EC-50 effects at 80 mM), this mechanism is likely to be more relevant to moderate oral alcohol consumption (20-30 mM brain concentrations). In ex vivo experiments we will determine the extent and distribution of ethanol modulation of extrasynaptic GABA-A receptor signaling. We will also test the effects of low doses of ethanol on the spontaneous action potential firing of VTA neurons, and test if the effects depend upon GABA-A receptor activity. We will also compare the motivational effects of intra-VTA ethanol in wildtype and delta subunit ko mice in order to probe the behavioral contribution of this ethanol target.

Past and Present ACGT-funded Projects


2012-2013Jun WangEthanol consumption and long-term potentiation (LTP) in the dorsal striatum
2012-2013Andrew KayserExamination of the effects of a delta receptor (DOR) agonist on human alcohol consumption
2011-2012Eric JorgensonAssociation study of alcohol use in diverse populations
2010-2012Elyssa MargolisVTA mechanisms involved in stress-induced ethanol consumption
2010-2011Kara LynchEffect of polymorphisms in UGTs and SULTs on the excretion of alcohol biomarkers
2009-2011Jennifer WhistlerDelta opioid receptor subtype-selective effects on ethanol seeking
2009-2010Louis PtacekNon-circadian behavioral assessment of mice carrying human clock mutations
2008-2010Frederick WolfPuckered and Decaptentaplegic signaling in ethanol behaviors in Drosophila
2008-2009F. Woody HopfDistinct atypical PKC isoforms in the nucleus accumbens and ethanol response


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