DEPARTMENT OF HEALTH AND HUMAN SERVICES
NATIONAL INSTITUTES OF HEALTH
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
151st Meeting of the
NATIONAL ADVISORY COUNCIL ON ALCOHOL ABUSE AND ALCOHOLISM
May 14, 2019
The National Advisory Council on Alcohol Abuse and Alcoholism (NIAAA) convened for its 151st meeting at 9:00 a.m. on Tuesday, May 14, 2019, at NIAAA headquarters in Bethesda, Maryland. The Council met in closed session from 9:00 a.m. to 9:38 a.m. to review grant applications and cooperative agreements. Dr. Abraham Bautista, Director, Office of Extramural Activities, presided over the Council’s review session, which, in accordance with the provisions of Sections 552b(C)(6), Title 5, U.S.C., and 10(d) of Public Law 92-463, excluded the public for the review, discussion, and evaluation of individual applications for Federal grant-in-aid funds. The closed session recessed at 9:38 a.m.
Council Members Present:
Carmen Albizu-Garcia, M.D.
Louis E. Baxter, M.D.
Howard C. Becker, Ph.D.
Jill B. Becker, Ph.D. (by telephone)
Daniel J. Calac, M.D.
Tom B. Donaldson
Alex M. Dopico, M.D., Ph.D.
Tatiana M. Foroud, Ph.D.
Robert J. Hitzemann, Ph.D.
Constance M. Horgan, Sc.D.
Scott J. Russo, Ph.D.
Arun J. Sanyal, M.D.
Vijay H. Shah, M.D.
Frank A. Sloan, Ph.D.
Susan M. Smith, Ph.D.
Edith Vioni Sullivan, Ph.D.
Constance M. Weisner, Dr.PH.
Karen Drexler, M.D.
Charles S. Milliken, M.D.
NIAAA Director and Chair: George F. Koob, Ph.D.
NIAAA Deputy Director: Patricia Powell, Ph.D.
Executive Secretary: Abraham P. Bautista, Ph.D.
Senior Staff: Lori Ducharme, Ph.D.; Ralph Hingson, Sc.D., M.P.H.; M. Katherine Jung, Ph.D.; George Kunos, M.D., Ph.D.; Antonio Noronha, Ph.D.; Megan Ryan, M.B.A.; and Bridget Williams-Simmons, Ph.D.
Other Attendees at the Open Session:
Approximately 35 observers attended the open session, including representatives from constituency groups, liaison organizations, NIAAA staff, and members of the general public.
Call to Order and Introductions
NIAAA Director George Koob, Ph.D., called the open session of the Council meeting to order at 9:50 a.m. on Tuesday, May 14, 2019. Council members and senior NIAAA staff introduced themselves.
Hughes Award Presentation
Dr. Koob presented the Senator Howard Hughes Memorial Award to Geoffrey K. Mumford, Ph.D., Associate Executive Director for Government Relations in the Science Directorate of the American Psychological Association (APA). Dr. Koob recognized Dr. Mumford’s efforts at the APA to promote research and the translation of evidence-based findings toward the goal of improving prevention and treatment of alcohol and other substance use disorders; his role in founding Friends of NIAAA and Friends of NIDA, and his spearheading of Congressional briefings about emerging research.
Dr. Koob highlighted key recent NIAAA activities, referring to the written Director’s Report, which was distributed to Council members.
Staff Transitions: Dr. Koob welcomed new NIAAA staff members Yoo Sun Kim, Ph.D., who joined the Laboratory of Molecular Signaling (LMS) as a Visiting Fellow in February 2019, and Nyamer Koat who joined the Division of Medications Development (DMD) as a Health Research Associate.
Budget: NIH received a total of $39.3 billion for Fiscal Year (FY) 2019. Of that, NIAAA received a total of $525.6 million, a 3.1 percent increase over the previous year. The FY 2020 budget is under development.
Funding Opportunity Announcements (FOAs): NIAAA recently issued two FOAs, one for Mechanistic
Studies on Chronic Alcohol Use and Sleep Homeostasis (R01) and one for Alcohol and Other Substance Use Research Education Programs for Health Professionals (R25). In addition, the following Notices of Special Interest were announced: Methodological Advances to Improve Alcohol Measurement and its Consequences for People Living with HIV who have Comorbidities, Coinfections, and Complications; Supporting Administrative Supplements for Fetal Alcohol Spectrum Disorders (FASD); and Development and Dissemination of Behavioral Treatments for Alcohol Use Disorder (AUD). Recently issued NIH-wide FOAs in which NIAAA is participating include, among others, two BRAIN Initiative opportunities (Secondary Analysis and Archiving of BRAIN Initiative Data [R01], and Tools to Facilitate High-Throughput Microconnectivity Analysis [R01]), as well as renewals for the Adolescent Brain Cognitive Development Study (Data Analysis, Informatics and Resource Center [U24], Linked Research Project Sites [U01], and Coordinating Center [U24]).
NIAAA Outreach: Several NIAAA staff members made presentations at the Community Anti-Drug Coalitions of America (CADCA) Leadership Forum in February 2019, including Dr. Koob, who described NIAAA priorities, research advances, and resources for prevention and treatment of alcohol use disorder; Ralph Hingson, Sc.D., who presented “Trends and Interventions that Work to Prevent Underage Drinking;” and Aaron White, Ph.D., who discussed “Alcohol and Opioids –A Deadly Combination.” NIAAA, along with the National Institute on Drug Abuse (NIDA) and the Substance Abuse and Mental Health Services Administration (SAMHSA), received the National Leadership Award from CADCA that recognizes significant contributions to the field of substance abuse prevention.
Advancing Technology for the Treatment and Prevention of AUD: In April 2019, NIAAA hosted a “Taking Stock of Advancing Technology for the Treatment and Prevention of Alcohol Use Disorder” workshop. The event included a panel of experts discussing topics such as applications of technology for research and clinical use, privacy and ethical issues, and barriers to adoption.
NIAAA Priorities for 2019: NIAAA has cross-Division teams working on the following: the Clinician’s Navigator (companion to the Alcohol Treatment Navigator); development of informational resources (Clinician’s Core Resource, Core Prevention Resource, and Core Liver Resource); FASD research guidelines; research on high intensity drinking, research on alcohol and aging, and recovery from alcohol use disorder; and mentor training to support diversity in NIAAA’s biomedical workforce.
NIAAA Outreach--Social Media Highlights: For one week in April 2019, NIAAA participated in a takeover of NIH’s social media accounts. The expanded audience resulted in increased reach for NIAAA’s messages. For Alcohol Awareness Month (April), NIAAA participated in Twitter chats on the topics of adolescent alcohol screening and FASD.
Research Highlights: Dr. Koob presented highlights of recent NIAAA-supported research:
“Biomarkers of Macrophage Activation and Immune Danger Signals Predict Clinical Outcomes in Alcoholic Hepatitis” was published in Hepatology (Epub 2019 Mar 19) by B Saha, D Tornai, K Kodys, A Adejumo, P Lowe, C McClain, M Mitchell, A McCullough, D Srinivasan, A Kroll-Desrosiers, B Barton, S Radaeva, and G Szabo. This study assessed a panel of recently identified potential biomarkers of tissue injury and immune cell activation as predictors of mortality and other clinical outcomes in alcoholic hepatitis (AH). Results revealed multiple new biomarkers to indicate AH severity and outcomes. Specifically, plasma levels of soluble cluster of differentiation 14 (sCD14; a host response indicator), soluble cluster of differentiation 163 (sCD163; a macrophage activation marker), and osteopontin (OPN; a phosphoprotein involved in neutrophil activation) were independent predictors of 90-day AH mortality, infection, and organ failure, respectively.
“Preconception Paternal Alcohol Exposure Exerts Sex-Specific Effects on Offspring Growth and Long-Term Metabolic Programming” was published in Epigenetics Chromatin (2019 Jan 22; 12:9) by RC Chang, H Wang, Y Bedi, and MC Golding. This study revealed multiple effects of chronic paternal alcohol exposure (prior to conception) on offspring that persisted into adulthood, including prolonged fetal gestation and growth deficits in males, sex-specific alterations in metabolic function, alterations in immune signaling, and increased pro-fibrotic structural proteins (markers of hepatic fibrosis) in adult male offspring. These abnormalities may suggest alterations in a sperm-inherited epigenetic program that influences the formation and function of the placenta. Importantly, these findings suggest that preconception lifestyle choices of biological fathers may impact offspring.
“Chronic Alcohol Drinking Slows Brain Development in Adolescent and Young Adult Nonhuman Primates” was published in eNeuro (2019 Apr 9; 6) by TA Shnitko, Z Liu, X Wang, KA Grant, and CD Kroenke. Alcohol misuse during late adolescence and early adulthood is a risk factor for the development of AUD. This study used a macaque model of daily alcohol self-administration and in vivo imaging to quantify the impact of chronic alcohol exposure on neural alterations that occur during this developmental period. The study found that 1) brain white matter growth is reduced in macaques identified as heavy drinkers in late adolescence, and 2) heavy ethanol consumption attenuates thalamic growth in the developing brain. These results demonstrate that heavy alcohol exposure during the transition to young adulthood significantly impacts brain development, an insult that may lead to the continuation of heavy drinking throughout later adult life.
“Inactivation of a CRF-Dependent Amygdalofugal Pathway Reverses Addiction-Like Behaviors in Alcohol-Dependent Rats” was published in Nature Communications (2019 Mar 18;10:1238) by G de Guglielmo, M Kallupi, MB Pomrenze, E Crawford, S Sierra Simpson, P Schweitzer, GF Koob, RO Messing, and O George. Investigators optogenetically inhibited corticotropin-releasing factor (CRF)-containing projections from the central nucleus of the amgydala (CeA) to four different brain regions. Results indicated that only inhibition of the CeA-bed nucleus of the stria terminalis (BNST) projection replicated previous findings of decreased drinking obtained with destruction of withdrawal activated neuronal ensembles in the CeA with Daun02.
“Endocannabinoid Control of the Insular-Bed Nucleus of the Stria Terminalis Circuit Regulates Negative Affective Behavior Associated with Alcohol Abstinence” was published in Neuropsychopharmacology (2019 Feb; 44: 526-537) by SW Centanni, BD Morris, JR Luchsinger, G Bedse, TL Fetterly, S Patel, and DG Winder. Using a mouse model of chronic alcohol consumption followed by forced abstinence, this study demonstrated that the endocannabinoid-sensitive projection from the insular cortex to the dorsal bed nucleus of the stria terminalis (dBNST) plays a key role in regulating negative affective behavior associated with abstinence from alcohol. These results establish the insula-dBNST neurocircuit as a promising target for endocannabinoid-based pharmacotherapy to alleviate negative affective symptoms associated with abstinence in AUD.
“Chronic Intermittent Ethanol Exposure Selectively Increases Synaptic Excitability in the Ventral Domain of the Rat Hippocampus” was published in Neuroscience (2019 Feb 1;398:144-157) by SE Ewin, JW Morgan, F Niere, NP McMullen, SH Barth, AG Almonte, KF Raab-Graham, and JL Weiner. Despite evidence for distinct functional roles of hippocampal subregions, the discrete effects of chronic alcohol on synaptic transmission in the ventral (vHC) compared to the dorsal hippocampus (dHC) have not been characterized. This study demonstrated that withdrawal from chronic intermittent alcohol exposure enhances synaptic excitability specifically in the vHC, providing insight into a neural mechanism that may contribute to the negative affect observed during abstinence in AUD.
“The IncRNA BDNF-AS is an Epigenetic Regulator in the Human Amygdala in Early Onset Alcohol Use Disorders” was published in Translational Psychiatry (2019 Feb 6; 9:34) by JP Bohnsack, T Teppen, EJ Kyzar, S Dzitoyeva, and SC Pandey. This study compared brain-derived neurotropic factor antisense (BDNF-AS), a long non-coding RNA that negatively regulates BDNF expression, and associated epigenetic mechanisms in the postmortem human amygdala in individuals with AUD who began drinking either before (“early onset”) or after (“late onset”) age 21 to age-matched control samples. Results revealed that the alcohol-induced epigenetic modifications of amygdala BDNF-AS impact BDNF expression and suggest an important role for BDNF-AS in the regulation of synaptic plasticity via epigenetic reprogramming in the amygdala of “early onset” AUD subjects.
“The Relationship between Impaired Control, Impulsivity, and Alcohol Self-Administration in Nondependent Drinkers” was published in Experimental and Clinical Psychopharmacology (Epub 2019 Jan 28) by CL Vaughan, BL Stangl, ML Schwandt, KM Corey, CS Hendershot, and VA Ramchandani.
Loss of control over drinking and impulsivity are key features of AUD. This intramural study examined the relationship between impaired control over drinking and alcohol consumption, and the modulation of this relationship by impulsive personality traits in drinkers without AUD in a human laboratory paradigm. Impaired control was associated with higher alcohol self-administration and positive urgency (the tendency to act rashly during positive mood states). These findings highlight the critical role of impaired control as a mediator of the relationship between impulsivity, alcohol consumption, and subjective responses in drinkers without AUD.
“Evaluation of Drinking Risk Levels as Outcomes in Alcohol Pharmacology Trials: A Secondary Analysis of 3 Randomized Clinical Trials” was published in JAMA Psychiatry (Epub 2019 Mar 13) by DE Falk, SS O'Malley, K Witkiewitz, RF Anton, RZ Litten, M Slater, HR Kranzler, KF Mann, DS Hasin, B Johnson, D Meulien, M Ryan, and J Fertig. A secondary analysis of data from three multi-site AUD pharmacotherapy trials was conducted to evaluate 1-and 2-level reductions in World Health Organization (WHO) drinking risk level as AUD treatment outcome measures. These measures capture reduction in drinking, an outcome which is more often achieved than outcomes currently accepted by the Food and Drug Administration (FDA), i.e., total abstinence or no heavy drinking days. Results suggest that WHO drinking risk level reduction is equally or more sensitive to treatment compared to FDA-accepted outcomes, demonstrating that this measure is an indicator of treatment efficacy that could be included as an additional outcome for AUD pharmacotherapy trials.
“DSM-5 Alcohol Use Disorder Severity as a Function of Sexual Orientation Discrimination: A National Study” was published in Alcoholism: Clinical and Experimental Research (2019 Mar; 43:497-508) by SE McCabe, TL Hughes, BT West, P Veliz, and CJ Boyd. This study examined the association between the Diagnostic and Statistical Manual for Mental Disorders (DSM)‐5 AUD severity and sexual orientation discrimination using a nationally representative sample. Discrimination associated with sexual orientation was associated with significantly higher levels of AUD severity, with proximal (past-year) experiences of discrimination more salient than more distal experiences, providing new evidence that sexual minorities who experience high levels of discrimination are at an increased risk of severe AUD.
Division of Neuroscience and Behavior (DNB) Concept Clearance
Changhai Cui, Ph.D., DNB, introduced two concept clearances: 1) Impact of Alcohol on Alzheimer’s Disease (AD) and Related Dementia, and 2) Neurobehavioral Mechanisms of Social Isolation and Alcohol Use Disorder.
Alcohol and Alzheimer’s Disease (AD) and Dementia: AUD is associated with a high risk of AD, as well as vascular dementia. However, the mechanisms contributing to these relationships are not well understood. The goal of the future FOA is to promote basic and clinical research to understand how alcohol influences susceptibility and progression of AD and related dementia. To achieve this goal, NIAAA proposes the following areas of research: Determine the differential effects of alcohol on neurocircuit specific pathology associated with onset and progression of dementia; examine how alcohol modulates neuroinflammation or neuroimmune interactions associated with pathologic aging and dementia; study how alcohol alters disease progression through changes in the epigenetic landscape of the aging brain; determine how alcohol’s effects on the neuroendocrine system contribute to neuropathological and cognitive functional changes associated with dementia; investigate sex differences in alcohol’s impact on vulnerability to develop dementia and progression of AD; study how neurovascular impairment plays a role in cognitive decline associated with chronic heavy alcohol use and how alcohol impacts disease progression through peripheral and central interactions; and determine how prenatal or adolescent alcohol exposure impact the susceptibility to and development of dementia. NIAAA will collaborate with the National Institute on Aging (NIA) on this initiative and leverage the studies supported by current Alzheimer’s disease (AD)/Alzheimer’s Disease and Related Dementia (ADRD) supplements. The proposed type of FOA is the Request for Applications (RFA) with set asides or a Program Announcement (PAR) with specific review criteria, using the R01/ R21 mechanisms. The anticipated outcome is a better definition of how alcohol impacts multiple neurobiological components that play critical roles in the onset and progression of the AD and related dementia.
Council members recommended broadening the scope to other age-related issues; animal models of AD and interactions with NIA scientists; prenatal alcohol exposure and human cohorts of those aging with FASD; damage on non-brain organs in the contribution to dementia; NIA AD/ADRD supplements; including the identification of differential and interactive neuropathological underpinnings of multi-facets of dementia; and individuals with a history of heavy drinking. In response, NIAAA will encourage alcohol investigators to collaborate with AD experts. Prenatal alcohol exposure studies on later-life dementia are within the scope of this RFA so it is appropriate to include those with FASD. Neurovascular impairments, the dysregulation of central and peripheral interaction, and studies on multi-facets of dementia are also within the scope of the RFA. Twenty-eight supplemental applications for FY 2018 and FY 2019 have been received.
Neurobehavioral Mechanisms of Social Isolation and Alcohol Use Disorder: There is strong evidence that social isolation affects brain circuitry in ways that interact with alcohol’s effects. The goal of this FOA is to call for integrative research studies of the behavioral, cognitive, and neurobiological mechanism(s) underlying the effect of social isolation on AUD. The scope of the FOA includes: Determining the extent to which neural systems responsive to social isolation interact with neural systems critical to pain perception and alcohol addiction, withdrawal, and relapse; understanding sex, aging, and individual differences in the impact of social isolation on alcohol use, relapse, and their underlying mechanism(s); identifying behavioral-or bio-markers linking social isolation and alcohol addiction that can be targeted for prevention; and identifying mechanisms underlying specific cognitive impairment(s) associated with alcohol addiction that can be reversed by improving social connections and support, or that may be exacerbated by social isolation. Research with animal models must demonstrate comparable and direct translational linkage to the understanding of and/or alleviating the impact of social isolation on human AUD. The expected outcome of this work is to fill a knowledge gap in the understanding of the etiology of AUD and co-occurring conditions such as negative affect and pain perception, and identifying potential novel interventions including social, behavioral, cognitive, and pharmacotherapy for AUD and related conditions.
Council members’ comments on this concept included: The focus of the proposal is too narrow; developmental impact and sex differences are important; social isolation needs to be carefully defined in animal studies; and an operational definition of “social isolation” is important to uncover causal effects. The scientific contact for this concept is Benjamin Xu, Ph.D.
Discussion: Howard Becker, Ph.D., recommended expanding the scope of the alcohol and AD concept to broader issues of aging, such as alcohol’s effect on bone density, the gut, and/or orthopedic pain. Dr. Koob interjected that NIAAA has a task force on aging that will address those non-cognitive issues. Dr. Becker also commented, in regard to the social isolation concept, that there is a disconnect in translating findings from animal studies to human ones. In rodents, social isolation has an impact on drinking when it occurs in adolescents, but this is more difficult to demonstrate in adult animals. In contrast, most clinical studies have focused on adults. Louis Baxter, M.D., supported NIAAA providing the scientific underpinning to Alcoholics Anonymous (AA)’s long-term contention that frequency of drinking is reduced when one is surrounded with others in recovery. Dr. Koob concurred, noting that the “deaths of despair” may be related to loneliness and social isolation. Frank Sloan, Ph.D., noted the challenge in studying the impact of prenatal alcohol exposure on AD in the absence of longitudinal data, expressing concern about the potential quality of the data in applications. Susan Smith, Ph.D., reported that dementia was the top health concern among those with FASD; this population appears to be aging more rapidly than others. She expressed strong support for the RFA. Edith Sullivan, Ph.D., commented that new methods such as neuroimaging hold promise for repurposing data to study FASD and aging. She noted that individuals with AUD have often been excluded studies on AD and Parkinson’s so alcohol’s impact on their development is unknown. Tatiana Foroud, Ph.D., reiterated Dr. Sullivan’s point, noting that research looking for biomarkers of AD excludes those with AUD. Constance Weisner, Dr.PH., agreed that this RFA is important, encouraging NIAAA to continue its emphasis on FASD and on women and drinking. She particularly noted the importance of understanding how to intervene in drinking among young women in their childbearing years. Tom Donaldson noted that the U.S. Centers for Disease Control and Prevention (CDC) reports that 10 percent of pregnant women use any alcohol and 3 percent engage in binge drinking; the 3 percent of binge drinkers account for 125,000 live births per year. Scott Russo, Ph.D., commented that the neuropsychiatric symptoms of alcohol use, e.g., aggression and anti-social personality, are completely understudied. He recommended that they be specifically mentioned in the RFA.
Division of Metabolism and Health Effects (DMHE) Concept Clearance
Kathy Jung, Ph.D., Director, DMHE, presented a concept clearance for multi-site observational studies of alcohol-related organ diseases. The goal of the proposed initiative is to verify the relevance of animal studies that lead to the discovery, development, and optimization of effective therapeutic and prevention strategies that improve human health and well-being, and to reduce the delay in disseminating the results. The rationale for this concept clearance is that alcohol-related organ diseases are a significant burden on health. Barriers to progress include limitations of animal models; insufficient knowledge of factors affecting risk, etiology, onset, and progression of diseases; and small patient populations that are dispersed over wide geographic areas. Research requires multi-disciplinary collaboration sharing research resources and patient populations. The purpose of the future FOA is to fund collaborative and coordinated prospective observational studies that will provide in-depth understanding of the natural history of alcohol-associated organ disease; provide insights to facilitate discovery efforts, and lead to new approaches to diagnosis, prevention, and treatment of organ damage due to alcohol. The proposed scope of the initiative is to conduct multi-site observational studies at three or more highly integrated and collaborative research sites with a shared study protocol and a single set of Specific Aims that can be completed in five years. Examples, but not limited to these topics are 1.) discovering biochemical and molecular links and risk factors for the onset of alcohol-associated organ diseases; 2.) identifying diagnostic and prognostic biomarkers and validating them in an independent cohort; 3.)optimizing processes of medical management leading to best clinical outcomes. An example of such a study is a prospective observational trial on early liver transplant. Current guidelines for a liver transplant require six months of abstinence, but this is not a reasonable requirement for those with advanced liver disease. Thus, it would be valuable to conduct research to 1) assess feasibility and efficacy of early liver transplants in cases of severe alcohol-associated liver disease (AALD); 2) assess processes for patient selection and medical management; and 3) determine long-term outcomes of liver transplant patients with AALD. This study would provide evidence that informs optimal selection for transplant and clinical management that leads to best long-term outcomes, and would evaluate the management of co-morbidities common to AALD, including AUD.
Council recommended including multi-site analyses leveraging regional differences in disease prevalence; using the advantage of sufficient power for discovery and validation of biomarkers; and using data-driven methods for analysis of the multi-faceted and multi-layered data, including machine learning. Council members commented that this research will facilitate genetic/environmental analyses of alcohol interactions; is well poised to achieve the proposed goals; and is a timely concept to enhance knowledge of alcohol’s overall impact on health and wellness so physicians can better advise patients and individuals can decide how much to drink.
Discussion: Arun Sanyal, M.D., praised the emphasis on verification of animal models because researchers can sometimes cure diseases in animals but fail to translate that success to humans. He recommended not funding phenotype studies that do not translate. Vijay Shah, M.D., pointed out that education of the public increases organ donation rates, but warned that the liver transplant field needs to be measured as it moves forward so that transplants are not expanded too rapidly and that patients who can actually benefit are those receiving transplants. Dr. Smith suggested that machine learning could be used in data-driven studies in which existing records are mined without having to recruit a human cohort. She also noted that researchers have over-relied on a single strain of mice, which has provided consistency across studies but limits the findings because it represents only one phenotype.
Brain Initiative Update
Dr. Koob introduced former Council member James Eberwine, Ph.D., who is NIAAA’s liaison to the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative.
Background: In June 2014, BRAIN 2025: A Scientific Vision outlined the goal of the Initiative: “… to map the circuits of the brain, measure the fluctuating patterns of electrical and chemical activity flowing within those circuits, and understand how their interplay creates our unique cognitive and behavioral capabilities.” The first five years of the Initiative, now completed, emphasized technology development; the second five years emphasize discovery-driven science. The overall budget for the Initiative is $5 billion. Priority areas include cell types, cell/circuit tools, human imaging/modulation, understanding circuits, training and dissemination, data coordination/informatics, and neuroethics. Funding has increased sharply in recent years, rising to $429 million for FY 2019.
Alcohol Investigators in BRAIN Initiative: Dr. Cui joined Dr. Eberwine to report on alcohol investigators and BRAIN Initiative projects. Over the past five years, alcohol investigators have been involved in seven studies. She highlighted two to illustrate this work. The first, by Zhenpeng Qin, Ph.D., and Paul Slesinger, M.D., focused on the development of new photo-releasable neuropeptide nano-vesicles for studying modulation in the brain. They developed a novel technique to regulate neuropeptide release in a time and dose dependent manner, and deployed an interdisciplinary team. In the second, NIH Post-Doctoral Fellow Miriam Bocarsly, Ph.D., mapped the neuronal circuitry underlying indirect striatal to hypothalamic connectivity and its role in feeding. This study was noteworthy for combining transgenic mice, viral tracing, designer receptors exclusively activated by designer drugs (DREADDs), and behavior analysis. It exemplifies BRAIN’s support for the next generation of investigators.
BRAIN Initiative 2.0: Dr. Eberwine presented a list of open FOAs, many of which NIAAA helped develop. Twenty percent of the total $5 billion has been spent, but the remainder will be allocated over the coming years. To expand the science given the progress to date, NIH Director Francis Collins, M.D., Ph.D., created an Advisory Committee to the Director (ACD) BRAIN Initiative Working Group “2.0.” The Working Group posted a report online for public comment. Its recommendations included: Bring cell-and circuit-based scales to human neuroscience; keep up the momentum of technology development; bridge scales; exploit the artificial intelligence (AI) revolution and analyses of natural behaviors; improve dissemination of new technologies and data sharing; improve diversity by recruiting scientists from related disciplines; and bring the public on board.
Neuroethics: Dr. Eberwine serves as co-chair of the BRAIN neuroethics subgroup established by Dr. Collins. To date, the Subgroup has produced a standalone Roadmap focused on neuroethics issues, and integrated neuroethics comments into the Working Group 2.0 report. These comments included increasing the BRAIN neuroethics budget from its current 1.8 percent level to 5 percent. The December 12, 2018 issue of the Journal of Neuroscience featured two articles about neuroethics and the BRAIN Initiative: “Neuroethics Guiding Principles for the NIH Brain Initiative” and “Neuroethics for the National Institutes of Health BRAIN Initiative,” of which Dr. Koob is a co-author with the other BRAIN IC Directors.
International BRAIN Initiative: There is an International BRAIN Initiative focused on global collaboration and knowledge sharing. It includes Australia, China, Canada, Korea, and Japan.
Updated Website: The NIH Brain Initiative website (www.braininitiative.nih.gov) has been redesigned, including an improved Funding Opportunities section and BRAIN Program pages.
Discussion: Dr. Koob inquired about the progress and direction of a European BRAIN project. Dr. Eberwine responded that there has been good progress and it is on track. However, there is still controversy on the aspect of circuitry that comes from the balance of actual biological experimentation versus just computational. Getting the right balance is problematic due in part to funding constraints.
Consideration of February 7, 2019 Minutes/Future Meeting Dates
Council members unanimously approved the minutes of the NIAAA Advisory Council meeting held February 7, 2019. Dr. Bautista announced upcoming meeting dates. In 2019, the Council will meet on September 12. Council meetings in 2020 will be held on February 6, May 12, and September 10; the CRAN Council will meet in 2020 on May 13. In 2021, the Council will meet on February 4, May 11, and September 9; the CRAN Council will meet on May 12, 2021.
Council broke for lunch at 11:50 p.m. and reconvened at 12:48 p.m. for the afternoon session.
Council Member Presentation: Understanding How Genetics Impact the Effects of Alcohol Throughout the Lifespan
Dr. Koob introduced Tatiana Foroud, Ph.D., to discuss genetic research on alcohol use disorder (AUD) and fetal alcohol syndrome disorders (FASD). She began by emphasizing the importance of phenotypes because diagnosis typically captures a dichotomous outcome, yet alcohol-related disorders are heterogeneous. She encouraged consideration of a range of phenotypes and collecting more detailed phenotypic/clinical information to allow for a wider range of analyses on specific aspects of disease to achieve a greater understanding of the underlying etiology and the development of therapeutics.
AUD: The goal of the Collaborative Study on the Genetics of Alcoholism (COGA), in which Dr. Foroud is an investigator, is to understand the role of genes, functional networks, and neurobiological and environmental factors on risk and resilience over the developmental course of AUD. Consisting of 11 study sites, the family study recruited alcohol-dependent probands from treatment facilities and used community-based comparisons. It recruited extended family members and conducted extensive multimodal phenotyping of them, including clinical, neurophysiological, neurocognitive, behavioral, and environmental/social characteristics. COGA families are ideal for genetic studies because they are large in number (17,762 subjects in 2,255 families) and many are densely affected with AUD; diverse (4,001 subjects in 575 African American families); and genetically informative (12,145 with GWAS [in dbGaP]). Diversity is important because phenotypes differ according to which subset of risk variants are inherited.
Looking at Genome-wide Association Study (GWAS) results, Dr. Foroud focused on the alcohol dehydrogenase 1B ( ADH1B ) gene because of its ubiquity and the range of phenotypes associated with it, such as the number of criteria in DSM-IV that are met (i.e., a measure of severity), desire to cut down, tolerance to alcohol, and severity phenotype. Self-Rating of the Effects of Ethanol (SRE) is an important phenotype in the COGA study. It asks subjects how they felt the first five times they took a drink, how they felt during the most recent three consecutive months, and during the period of their heaviest drinking, across a continuum of four potential effects from none to passing out. A total SRE score is then generated for each subject. COGA analyses revealed a relatively strong genome-wide finding of total SRE in both its European-American and African-American subsets. These findings were compared to those in NIAAA samples, where the effect was replicated in the African-American sample, but not the European-American one, and in a sample of European-Americans in San Diego where the finding was again replicated. Thus, findings were not consistent across studies using different sampling strategies, as would be expected for a complex and heterogeneous disorder like AUDs and other alcohol-related phenotypes.
COGA is conducting a longitudinal prospective study using a sample of offspring between the ages of 12-22 to look at developmental trajectories based on bi-annual multi-modal assessments. A particular strength of COGA is the ability to test the influence of genetic findings as they unfold during adolescence and young adulthood and in the context of environment. COGA incorporates three emphases: genetics, lifespan, and brain function. Age and sex-specific differences were also noted, as well as differences in reward processing. Cellular modeling to explain the variants is desirable, e.g., effects have been seen in analyses of the potassium voltage gated channel subfamily J member 6
(KCNJ6) gene. But even in ideal cases where the researchers know exactly what they want to study on a specific gene in a region, they still struggle to understand how to use this information to inform the phenotype and development of a related intervention targeting AUD. More common in genetic studies is the struggle to understand how to use the genetic associations that are identified and interpret them with increasing amounts of data from other sources to understand their meaning. For example, COGA found an association with chromosome 1(CHR1) located on Chromosome 17, in a region encompassing 13 genes. Integration of expression quantitative trait loci (eQTLs) narrowed the study down to six genes, and conditional analyses prioritized two of them. Splicing QTLs and methylation QTLs (meQTLs) allowed the researchers to prioritize one gene that led COGA investigators to focus on corticotropin-releasing hormone receptor (CRHR), which had been previously linked to alcohol preference in rodent models. Dr. Foroud stressed that understanding what contributes to variation and increasing annotation is essential. Increasingly, researchers need to think not just about associations, but about associations within subsets and what they mean.
FASD: Dr. Foroud also presented information about the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD). Its purpose is to inform and develop effective interventions and treatment approaches for FASD, through multidisciplinary research involving basic, behavioral and clinical investigators and projects. As with the COGA studies, the theme in this area is that classification cannot simply be dichotomous. Instead, CIFASD uses a multidisciplinary approach to study human FASD, encompassing dysmorphology evaluation, neuropsychological assessment, three-dimensional (3D) facial imaging, magnetic resonance imaging (MRI), and demographic information. The analysis strategy is to compare genetic variants between subjects exposed to prenatal alcohol use with and without FASD.
It is important to identify genetic variants that confer protective effects reducing the risk of FASD in the context of prenatal alcohol exposure, as well as variants that increase the risk for features of FASD in the presence of prenatal alcohol exposure. In preliminary analysis, variants in the Cysteine Rich PAK1 Inhibitor (CRIPAK) gene, a cystine-rich PAK1 Inhibitor, was associated with an increased risk for FAS. Individuals with prenatal exposure but without features of FAS did not carry rare variants in CRIPAK.
The challenge in doing increasingly more rigorous work in this area is sample size. One strategy for addressing this problem is to develop a better way to identify the phenotype. CIFASD’s approach is to examine the face, initially with 3-D and now with inexpensive 2-D imaging (i.e., an iPhone photo), turning it into a quantitative trait. This approach allows individuals to be placed along a spectrum, rather than yielding a dichotomous outcome. In this “face signature” strategy, the goal is to compare an individual’s face to an average face to determine if he or she has features that are consistent with prenatal alcohol exposure. CIFASD research established that those individuals with facial features more closely associated with someone exposed to prenatal alcohol were also likely to have lower cognitive performance, and vice versa. Thus, this facial signature strategy is promising as a simple and less expensive way to identify those likely to have FASD and has allowed the field to assess more individuals. Dr. Foroud is trying to leverage this face signature strategy to increase the number of study participants by recruiting on social media and enrolling individuals through an online consent and protocol. Data collection includes saliva for DNA, prenatal alcohol exposure data, and facial photos.
Discussion: Dr. Koob inquired if there are other 3-D imaging studies in progress. Dr. Foroud replied there was a previous project that was a predecessor to the current CIFASD; its investigators are now part of CIFASD. Dr. Koob asked about the necessary sample size for the facial signature technique. Dr. Foroud responded developmental age and diversity are two important factors. Most researchers would estimate at least 50 to 100 control faces and at least 200 of those exposed to prenatal alcohol to address the full spectrum of FASDs. Dr. Sullivan asked if there is any aging out of FASD facial differences. Dr. Foroud reported that some characteristics may become subtler, but the basic aspects remain. Dr. Koob asked if Dr. Foroud thinks that NIAAA would be justified continuing work overseas in countries with a high prevalence of FASD. She responded affirmatively. Carmen Albizu-Garcia, M.D., inquired about the high number of research sites in the eastern and southeastern areas of the United States and whether Native Americans were being recruited. Dr. Foroud explained that the map of research sites she presented was specifically for the COGA study. She noted that CIFASD has encountered challenges in conducting research involving certain Native communities. Some were concerned about being photographed, and some tribes chose not to participate. Dr. Koob commented that NIH has agreements pending with the Navajo Nation about their participation in certain research studies. The agreement has been controversial because a website about the research was planned and some tribal members were concerned about online protection of research information. Dr. Calac stated that obstacles in recruiting Native Americans occur because of previous negative experiences. He noted that photographs and genetic information are particularly problematic and pointed to the need for community education about why it’s important to collect this data, which is happening now among the larger tribes. Dr. Foroud said that COGA researchers are hoping that the information they can offer back to the community will be a motivator for participation. Dr. Sanyal inquired if the facial recognition approach could be applied to all histological findings, expressing frustration that pathologists provide dichotomous diagnoses and valuable information is unavailable to the physician. Dr. Foroud responded that there is growing interest in matching data science with pathology to provide more nuanced information.
Council Member Presentation: Alcohol and BK Channels: Fundamental Mechanisms, Cerebrovascular Pathophysiology, and Discovery of New Drugs of Therapeutic Potential
Dr. Koob introduced Alex Dopico, M.D., Ph.D., who presented research that the University of Tennessee Health Science Center (UTHSC) Department of Pharmacology is conducting to develop small molecule agents to counteract disruption of physiology caused by alcohol intoxication via the identification of new targets (protein receptors) and associated mechanisms that mediate alcohol (ethanol)-induced disruption of physiology. One of the targets of interest is the BK (“big potassium”) channel, a homotetramer of slo1 proteins (so called BK α subunits) and their closely-associated regulatory proteins (so called BK β subunits). Slo1 proteins can go from closed to open states, with the equilibrium from the former to the latter being favored by positive membrane voltage (i.e., depolarization) and/or increased intracellular calcium binding to the cytosolic regions of this protein. If the BK channel is activated, there is a reduced release of neurotransmitters, decreased neuronal excitability, and vasodilation, among other cell physiology changes. If the channel is blocked, cerebral arteries constrict.
Physiology: The earliest study of alcohol and the BK channel sought to investigate the molecular bases of why people urinate after they drink alcohol. Alcohol reduces the release of anti-diuretic hormones, possibly due to an increase in BK channel function. To test this hypothesis, investigators isolated the nerve terminals from the neurohypophysis in rat studies. They discovered that ethanol indeed increased BK currents in the neurohypophysial terminals. They explored whether potassium channels other than BK were modulated by alcohol but found them totally resistant, contrary to the hypothesis that alcohol may be acting non-specifically on the lipids around the potassium channel receptors. The researchers then cloned the BK channel from mammalian CNS neurons and inserted it into an exogenous protein factory, the Xenopus laevis oocytes, in order to study BK-alcohol molecular interactions. They found that the effects of alcohol were very similar in both mammalian and amphibian systems, which differ dramatically in lipid composition. The concentration of alcohol needed to achieve the increased BK current was within the range of alcohol intoxication (10-100 mM).
The next question was: Why does the BK current go up in presence of alcohol? The researchers found that it increases only by shifts from the closed to the open state, which occurs because alcohol increases the affinity of BK for calcium. What they didn’t know, however, is where the alcohol goes; whether it’s binding inside of the slo1 protein itself, membrane surrounding lipid domain, or in nearby proteins that are closely associated with slo1. To solve this problem, the investigators cloned the protein and inserted it into an artificial lipid membrane. In that setting, alcohol still activated the BK channel cloned from human brain. Therefore, the alcohol-recognition site must be in slo1 itself or at the slo1-lipid. To find a binding site for a tiny ligand like ethanol in slo1, a protein with more than 1,200 amino acids, researchers faced a “David and Goliath” situation. Their starting point was to note that most voltage-gated ion channels in the superfamily of six transmembrane were resistant to alcohol at legal intoxication levels. They focused their search for an alcohol binding site in areas that were unique to the slo1 channel. These are important because these are the regions where calcium and magnesium bind, and alcohol’s action is related to calcium affinity. Further experimentation revealed eight regions where alcohol putatively could bind. Next, based on crystallographic information, researchers built a computational homology model for an ethanol-binding site in slo1 proteins in one of those eight regions. They found that only “region 2” (the second from the slo1 amino end) could hold an alcohol molecule. Further computational modeling based on crystallographic data indicated that K361, R514, E354, S357 and N358 within this “site or region 2” (aa. 354-367) were critical for ethanol interaction slo1. To verify the model, the researchers conducted multiple experiments using point mutations to alter the chemical bonds and the chemical properties of the surrounding components and determine the consequences of these changes on alcohol interaction with slo1 channels. They concluded that their computational model was successful because ethanol action was abolished by amino acid substitutions that prevented alcohol from H-binding to the site, disruption of chemical features of the site, and prevented ethanol access to the site. Further, they established that the alcohol-sensing area in the BK channel has defined dimensions. Finally, most importantly, the model explained previously reported phenomena: the cut-off for n-alkanols’ modulation of BK channels and the ligand (Ca2+)-dependence of ethanol action.
Behavior: Researchers at the University of California San Francisco (UCSF) studied the effects of alcohol on Caenorhabditis elegans, a free-living transparent nematode. They found that the T381I SLO-1 mutation had negligible effect on BK channel-driven behaviors such as motor behavior and egg laying themselves, but conferred strong resistance to ethanol intoxication. Transgenic expression of T381I or T352I rescued basal behavior but not ethanol intoxication in SLO1 null mutants. The slo-1 mutations considered in the UCSF studies were in the same region that the UTHSC scientists identified as alcohol-binding site in slo1 proteins from mammals. Looking at mammalian behaviors, the UTHSC scientists recorded trains of action potentials from medium spiny neurons (MSN) in mouse dorsal striatum slices before and after <5 min of in vitro ethanol perfusion in physiological saline because the action potential in that area is BK-dependent. The results showed that ethanol shut down the firing of neurons in the wildtype mice, which is the expected response to alcohol in such brain region. In contrast, neurons from a knock-in mice where the key amino acid-binding ethanol had been substituted, were alcohol-resistant. The results underscore the importance of a single amino acid in the mammalian slo1 protein in alcohol’s disruption of mammalian brain physiology. Moreover, experiments on mammalian behavior at La Jolla-Scripps using a two-bottle model reported that reduced alcohol sensitivity of BK is associated with slower drinking escalation in the knock-in when compared to wt mice.
Dr. Dopico concluded that BK channel-forming slo1 proteins are alcohol receptors that participate in alcohol-disruption of mammalian brain physiology and behavior.
Development of Therapeutics: The next question the UTHSC group approached was whether the human receptors in regard to the BK channel-forming slo1 protein-alcohol interaction is sufficiently similar to mice and worms and thus lead to development of therapeutics. To that end, the researchers studied the proteins in eleven species, from worms to humans. Their goal was the characterization of the ethanol site in human slo1 for pharmacophore design and eventual screening of novel agents that counteract slo1-driven alcohol actions. In comparisons of worms and humans, they found that ethanol (EtoH)-slo1 interactions occurred in topologically similar regions and that mslo1 Lys 361 is conserved in both species, acting as an H-bond donor. Where the species differ, however, is that mslo 1 Ser357 is conserved in humans and acts an H-bond donor, while in Caenorhabditis elegans, glutamate plays this role. These new findings are the stepping stone for the next challenge: develop small agents to block alcohol action on CNS neurons via BK channels.
In addition to its effects on neuronal function and behavior, moderate to heavy episodic alcohol intake contributes to events such as systemic hypertension, ischemic stroke, cerebral ischemia, transitory ischemic cerebral attack, hemorrhagic stroke, and mortality from stroke. Binge drinking can increase angina and the risk for cerebrovascular spasm. Despite the widely known effect of alcohol as a peripheral vasodilator (as its metabolite acetaldehyde dilates skin vessels), there is overwhelming evidence that alcohol constricts arteries, cerebral in particular. In experiments with rats, In vivo ethanol administration constricted the middle cerebral arteries (MCA) by ten percent in diameter, creating hypoxic conditions for the brain as MCA is the major blood provider to the brain. To determine if a circulating agent could prevent this constriction of the brain vessels, further studies with rats were conducted in which the MCA were isolated. The researchers established that ethanol action is independent of alcohol metabolism, as well as circulating and endothelial factors. Therefore, the target of the alcohol must be in the smooth muscle, which comprises the vast majority of the arterial mass. Knowing that BK channels are critical mediators of arterial diameter, the researchers looked at blood flow in the presence and absence of blockers. They found that BK channels are major mediators of ethanol-induced, endothelium-independent MCA constriction.
A review of the literature indicated that alcohol activates almost all BK channels from central neurons and the endocrine systems but not those from the arteries, which are inhibited following brief exposure to clinically relevant concentrations of ethanol. To understand this, the researchers found that all BK channels have associated, small regulatory sub-units. The β1 subunits are very abundant in vascular smooth muscle, while β4 subunits are abundant in the central nervous system (CNS) neurons. This raised the speculation that β1 subunits were necessary for alcohol to inhibit BK channels. Thus, the researchers studied β1 subunits in knockout mice and controls. They learned that in the presence of β1 subunits, ethanol inhibits BK currents in cerebral artery myocytes and evokes artery constriction whereas these actions were lost in the knockout mouse. Next, the investigators studied the effects of alcohol on all possible combinations of BK channels. They identified two phenotypes--potentiation (slo1; slo1+β3; slo1+β4) and inhibition (slo1+β1; slo1+β2)--using a strategy of supplementing tissues from β1 knockout mice with cDNA constructs. They found that alcohol’s effects on smooth muscle tissue were inhibited among the knockout mice loaded with exogenous β1 and among the mice with β4 TM2. Middle cerebral arteries were constricted among the mice with exogenous β1 but recovered the constriction with the exogenous β1 and β4 TM2 combination. Thus, it is apparent that β1 TM2 mediates ethanol-inhibition of smooth muscle BK channels and middle cerebral artery constriction.
Can scientists take advantage of these findings? Ongoing experiments at UTHSC revealed that the TM2 for β1 contains a binding site for endogenous lipids that the liver makes, i.e., bile acids. One that binds perfectly at the site is lithocholic acid (LCA). It binds to threonine, which is only found in β1 TM2. From that, they created pharmacophores, chemical structures that exactly sit on the predicted site. They found pharmacophores for agonists, i.e., vasodilators, and antagonists, theoretically vasoconstrictors. To determine if they reverse alcohol action, the researchers compared the impacts of Celastrol and LCA on artery diameter following administration of 50 mM of ethanol. The two compounds were successful in reversing constriction. They also studied this effect in vivo with similar findings. Thus, preliminary results suggest that β1 TM2-targeting agents that activate BK channels blunt MCA constriction by alcohol. This provides a platform for developing more potent drugs to antagonize alcohol-induced artery constriction.
Discussion: Dr. Shah posed a question about the heterogeneity of the effects of alcohol on different cell types, such as hepatocytes. Dr. Dopico responded that science first needs to understand which tissues in the body have critical functions that are BK determinant. At this time, it is unknown what the physiological effects are of BK in the hepatocyte, as well as in the heart and the endothelial cells. It is known that circulation in the liver (but not the hepatocytes) contains BK channels which are sensitive to both cholanes and alcohol. Dr. Howard Becker inquired if the compounds that block constriction of the arteries have behavioral consequences. Dr. Dopico responded that behavioral effects have not been tested; the researchers are in the earliest stages of identifying drugs that may have a physiological effect. But this is an important question. If BK channels are in every tissue, how can scientists target alcohol in one tissue without having a dysfunctional effect elsewhere? β1 is located in only a few places in the brain so it should be possible to target it directly to counter vasoconstriction without affecting other BK neurons. However, this needs to be proved. Dr. Sullivan asked if there is any protective value in having the vasoconstriction from alcohol. Dr. Dopico responded with a larger question: is there any evolutionary pressure to alcohol’s effects from invertebrates to humans? UTHSC scientists have studied alcohol’s effects across 11 species with similar results. Some species are particularly resistant, e.g., cows for whom ethanol is a protective mechanism. But the fundamental answer is unknown. Jill Becker, Ph.D., asked if there were sex differences in the effects of alcohol on the BK channel. Dr. Dopico responded affirmatively, noting that adult females constrict less so they are more protected from the effects of alcohol. Dr. Sanyal wondered if the BK channels evolved to handle endogenous or exogenous alcohol and if so, do they play a role in normal metabolic reprogramming of different cell types in response to the local milieu? Dr. Dopico responded that he didn’t know, pointing out that the channels have been advantageous from an evolutionary perspective because they helped slow the system down during periods of intense excitability.
Council Member Presentation: A Multi-Species Approach to Understanding Excessive Ethanol Consumption
Dr. Koob Introduced Robert Hitzemann, Ph.D., who presented research from Oregon Health and Science University (OHSU) and other sources on excessive alcohol use among different species. Dr. Hitzemann pointed out that such studies are important because they are translational and inform research on humans. Unlike in other fields where the validity of animal studies has been controversial, alcohol-related studies from pre-clinical to clinical have fit together fairly well.
Background: Research on excessive ethanol consumption addresses three areas: risk, individual variation, and the effects of chronic exposure. The goal is to find the “sweet spot” where the three areas overlap. Animal models allow scientists to dissect each of these areas, which is difficult to do in human studies. OHSU has conducted research with mice, rats, and rhesus and cynomolgus macaques. Dr. Hitzemann’s discussion of rodent data will focus primarily on selection. The selection phenotypes include preference and binge consumption. Preference references the 24/7 two-bottle choice design. The binge consumption phenotype is studied via the “drinking in the dark” approach in which, four hours after the dark phase, the animal’s water bottle is replaced with one with 20 percent ethanol. With selection, the researcher can achieve consumption of three-times the intoxication level. Individual variation has been particularly stressed in recent studies; some individuals from a positive family history simply don’t drink, in both humans and animals. Why? Of interest to researchers are the molecular mechanisms associated with each phenotype. The extra cellular matrix (ECM) and the cell adhesion molecules emerge repeatedly as related to excessive ethanol consumption in these studies.
Dr. Hitzemann’s lab’s approach is network-centric, i.e., looking at networks that are common across both animal and human models in search of hub nodes that could be targets for drugs. Looking at the evolution of species from mice to macaques to humans, Dr. Hitzemann stressed that the mouse is at least ten times more genetically diverse than any human population. Macaques are also very genetically diverse compared to humans. At the sub-cortical level, the brains of mice, macaques, and humans have similar functions. The central nucleus of the amygdala functions the same across the three species. The connections to the cortical areas are different because they’re more pronounced in humans. By focusing on areas of greatest similarity, researchers are most likely to find congruent results.
Methodological Approaches: OHSU has been using RNA-sequencing (Seq) data for over a decade to look at the transcriptome of its animal models. They have established that one can use RNA Seq to make gene networks. OHSU has also studied alternative splicing, which is important in the brain and has been understudied. Synaptic genes have the most alternative splicing; alternative splicing is likely to change gene function. Further research by Dan Iancu, Ph.D., mapping vectors of exon-mapped RNA Seq Data reads revealed there are large cosplicing networks in the brain. Selection for chronic excessive ethanol consumption affects those networks.
Selection: Much of the work in the alcohol field has been conducted with the B6 (C57BL/6J) mouse, somewhat less with its genetic cousin, the D2 (DBA/2J). Dr. Hitzemann’s lab bred heterogeneous stock collaborative cross (HS-CC) mice, which included the B6 and the PWK, both of whom are excessive ethanol consumers and provide high genetic diversity. Some years ago, OHSU also put together another 8-way heterogeneous stock; their selection had nothing to do with alcohol. These are the mice that were used in the high drinking in the dark (HDID) experiments conducted by John Crabbe, Ph.D. In these studies, selection was based on blood ethanol concentration, not on the amount the mice drank. Dr Crabbe did two selections, the second one (HDID-2) two years after the first (HDID-1). Dr. Crabbe was able to raise blood ethanol from low levels (10 mg/ml) to high levels (87 mg/ml) very quickly. Both lines now consume 200 mg/ml on average. In another study led by Dr. Iancu, these animals were genotyped. The expectation was that since Dr. Crabbe had selected the animals both times under the same conditions, that the mice would be the same. Instead, the two strains turned out to be very different; their genes moved in different directions. Looking more closely, Dr. Crabbe discovered that the HDID-1s were sippers and the HDID-2s were gulpers, so they drank alcohol differently, although they both reached the same blood alcohol concentration. Subsequently, they compared the HDID-2 mice to their founders, examining genes that are differentially expressed in males and females. There are major sex differences in the selection. In the males, there are many annotations associated with the ECM and in particular the collagens In both males and females there was a strong association with immune system genes. The focus here is on risk for excessive consumption and not ethanol consumption per se. The immune system genes affected are both pro- and anti-inflammatory genes. Thus, the researchers suspect that there is critical balance between pro- and anti-inflammatory genes that affect ethanol consumption.
Individual Variation: Among the HDID-1 and HDID-2 mice, it’s clear that the mean intoxication level is significantly above the commonly accepted definition of 80mg/ml but in both lines, there are animals that are not drinking. To determine why, the researchers took some of the HDID-1 animals and sequenced their transcriptome, analyzing males and females separately. In the females, the genes that were associated with increased consumption were synaptic. In males, there was a much stronger negative correlation with the ECM and positive correlations with the signaling genes. When male and female data were merged, the immune response annotation and chemokine activity were strongly involved, positively correlated with animals at risk for excessive consumption.
Macaque Studies: In a study of chronic ethanol exposure in cynomolgus macaques, the animals were biopsied in Area 46, the dorsolateral prefrontal cortex, before and after ethanol exposure. The findings were reminiscent of the mouse studies: In the animals that drank, there were changes in the immune response, cell adhesion genes, GTPase regulator activity, and ECM. In the immune category, the observed genes included both pro- and anti-inflammatory ones which were down-regulated. Looking at areas of the brain, the researchers observed that the immune response genes were down-regulated in all the brain regions. OHSU investigators have also looked at the effects of chronic ethanol exposure on rhesus macaques. Examining results in the central nucleus of the amygdala and Area 32, changes were seen in cell adhesion and post-synaptic density genes, as well as genes associated with glutamate function. This study did not, however, show a change in immune response system genes.
Rodent Studies: In further selection study for High and Low ethanol preference (HS-CC mice founders), RNA-Seq data were collected from the CeA, the nucleus accumbens shell (NAcS) and the prelimbic cortex(PL). A group of genes, the cadherins and protocadherins, emerged as highly associated with selection. These are the molecules that tell neurons where to grow in the brain. It is not clear what they do in the adult brain, although they are very highly expressed. Another gene that emerged in this study is the synaptic gene Dlg2, which encodes for PSD93 that is involved the regulation of N-methyl-D-aspartate (NMDA) receptors. Scientists have observed the important role of this gene multiple times, suggesting it might be a good target for modulating ethanol consumption. In another study using the HS-CC mice who were allowed to drink two-bottle choice for 13 weeks, the consumption patterns were highly variable between week 1 and week 13; consumption generally plateaued after 8 weeks. This suggests that conducting experiments of only one or two weeks in length may be producing misleading results. The animals were sacrificed after 13 weeks and their genes examined. Across all weeks, genes in the extracellular space in females were associated with high consumption; at week 13, genes involving the cilium organization were involved. Overlapping those two categories for females were cell adhesion, regulation of cell motility, actin binding, and the extracellular region. Immune response genes were not seen in this experiment, which appears to be consistent with the two-bottle model and different from the drinking in the dark design. This may be a difference in the phenotypes that needs to be kept in mind in future studies.
The Cilium as a Target: Emerging from the 13 week chronic studies was the strong effect on the primary cilium. Every cell has a primary cilium, which has some very unique proteins, but no glutamate receptors. The question arises as to whether modulating cilium function would affect ethanol consumption. Adenylyl cyclase type 3 (Adcy3) is one of the proteins unique to the primary intra-cilium proteins that could be targeted. Specific agonists and antagonists exist.
Human Studies: Dr. Hitzemann briefly reviewed data from human studies conducted by the University of Texas at Austin using data from the New South Wales Brain Bank. Across brain regions, these investigators found immune response gene categories to be highly associated with ethanol consumption. This finding is consistent across species; within the rodent population, it may be necessary to examine the preference vs. binge consumption data separately.
Conclusion: Looking across the domains of preference, binge drinking, and individual variation, it appears that some responses to alcohol are true across species and some are fairly unique. There remains a great deal to learn about alcohol’s effects across and within species.
Discussion: Dr. Koob identified a theme from Dr. Hitzemann’s presentation, i.e., that the more people drink, the more they are engaging their immune system. Dr. Hitzemann confirmed this applies to a higher concentration of alcohol in a shorter amount of time. Dr. Howard Becker commented on the challenges of studying neuroimmune genes in the CNS using polymerase chain reaction (PCR) assays because the genes express at very low levels; he was surprised that Dr. Hitzemann had not been able to find them using highly sophisticated tools such as RNASeq. Dr. Hitzemann said that a very large number of RNASeq reads is needed to study genes with low expression, such as the immune-related ones. The large number of reads also allows the scientists to assemble the alternative splicing data in a way that was not previously possible. Dr. Becker noted that the high concordance of outcomes with mouse and non-human primate models is an important validation of doing work with animal models. Dr. Russo inquired if anyone has sequenced the immune cells themselves since what might be happening is a homeostatic down-regulation of the system because the inflammatory signal is coming from outside of the CNS, i.e., looking at the issue from a core proteomics approach. Dr. Hitzemann responded that, to his knowledge, the answer is no. As sequencing technology becomes more accessible, the ability to do single cell work will become increasingly possible, as will the ability to analyze specific tissues and brain regions. Dr. Smith commented that one of the set of genes that Dr. Hitzemann didn’t address in his presentation was the ribosomal protein. They have often been dismissed but in her lab, they turned out to be highly mechanistic. Dr. Hitzemann replied that the ribosomal proteins are associated with mitochondria rather than the nucleosome.
Department of Veterans Affairs (VA): Karen Drexler, M.D., reported she is National Mental Health Program Director for Substance Use Disorders at the VA that provides care for six million veterans each year. The VA does a good job of screening for alcohol use and providing brief interventions and follow-up. She would like to expand the use of medications for treatment of AUD, but it’s a “heavy lift” for the VA’s primary care providers who lack a solid educational foundation about AUD. Therefore, she appreciates NIAAA’s efforts to educate the public and providers. Under the VA MISSION Act starting in June 2019, veterans will enjoy greater access to local care in the community and VA may have more authority to help direct them to good treatment. NIAAA’s Treatment Navigator may help in this effort. Another clinical priority at the VA is improving quality of care via measurement-based care so that veterans who start on treatment are assessed using the Brief Addiction Monitor developed by the VA and then re-assessed, which prompts clinicians to think about six evidence-based risk factors and six evidence-based protective factors for recovery and to address those factors in treatment planning. She believes this approach is the future in trying to tailor treatment to individual needs.
Department of Defense (DoD): Dr. Milliken reported that the Army has approved a voluntary care track in which a soldier can obtain confidential care for an AUD, in contrast to the previous policy which required clinicians to report alcohol-related care to the individual’s commander and to enroll the patient in a personnel database to track alcohol misuse, leading to expulsion from the service after the third report. He also acknowledged the contributions of Dr. Weisner who served on the DoD/Institute of Medicine (IOM) committee that recommended the new policy. The hope is that this new approach will now go DoD-wide.
There were no public comments.
Dr. Koob adjourned the meeting at 3:15 p.m.
I hereby certify that, to the best of my knowledge, the foregoing minutes are accurate and complete.
George F. Koob, Ph.D.
National Institute on Alcohol Abuse and Alcoholism
National Advisory Council on Alcohol Abuse and Alcoholism
Abraham P. Bautista, Ph.D.
Office of Extramural Activities
National Advisory Council on Alcohol Abuse and Alcoholism