Alison Poulson
Effects of alcohol addiction on DNA methylation in the amygdale
Abstract
One factor that makes alcohol addiction difficult to overcome is the anxiety that comes
during withdrawal. Research in mice has shown that histone deacetylation in the amygdala is
related to the symptoms of anxiety during ethanol withdrawal, and that drugs to reduce
deacetylation also reduce the amount of anxiety. If this pattern also applies in humans, it will
provide possible sources of drug therapy for individuals in withdrawal. This study will use the
LUMA method of measuring histone methylation in brain tissue from alcoholics suffering from
withdrawal at the time of death to determine whether similar patterns of chromatin remodeling to
mice are found in human alcoholics.
Introduction
Alcoholism - addiction to the consumption of ethanol - is a major health concern (Li et al
2004). The genetic processes underlying alcoholism and other forms of addiction are complex
and not completely understood. Addiction has an unusual effect on the brain because changes in
brain function are observed long after the stimulus is removed (Hyman et al 2006). Even if the
person has not had the addictive substance for years, a slight trigger can bring back all of the
physiological effects of the full-blown addiction. This trigger need not be the drug itself; a place
or person or thought associated with the addiction can cause the cravings to return.
How any stimulus can cause such long-term changes in brain function has been the focus
of much research in the past decade. Scientists have posited epigenetic mechanisms, or changes
in histone structure, as one of the causes (Renthal and Nestler 2008). DNA is associated with
histones in the nucleus, because histones provide organization for the DNA. The DNA strand is
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wrapped around histones and compacted to allow a long strand to fit into a small part of a cell
(Tsankova et al 2007). In the process, the DNA is compressed and packed, more in some parts
than in others. The less compact areas are easily reached by rRNA and often transcribed. More
dense regions, on the other hand, are transcribed only rarely or never.
The density of the DNA is determined by acetylation and methylation of the histones
(Tsankova et al 2008). The two processes work in tandem; acetyl groups are removed and
methyl groups added, packing the DNA more closely and precluding transcription of the DNA at
that point, or methyl groups are removed and acetyl groups are added, allowing the genes in that
region to be transcribed (Eden 1998). Though once it was believed that acetylation and
methylation was determined in utero and that no changes to chromatin structure were made after
birth, recent research has shown that chromatin remodelling is a normal occurance in the genome
(Tsankova et al 2008). Proteins have been identified that mediate the acetylation, deacetylation,
methylation, and demethylation of histones in response to cellular stimuli.
Drugs of addiction, including ethanol, are believed to affect the remodeling of the
chromatin (Pandey et al 2008). This change in structure gives rise to both the effects of chronic
alcohol consumption and the symptoms of withdrawal when ethanol consumption is stopped.
Specifically, anxiety is common in those who have recently stopped a chronic alcohol habit
(Pandey et al 2003). This anxiety is a type of negative feedback that makes a return to alcohol
consumption more likely. Researchers have posited that if these anxiety symptoms could be
mitigated, it would be easier for individuals to give up unhealthy alcohol habits (Pandey et al
2008).
One important area of research into the persistence of anxiety symptoms has been done in
mice. Researchers have identified proteins involved in histone acetylation and deacetylation that
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change activity levels based on ethanol consumption (Pandey et al 2003). Chronic ethanol
consumption in mice increases histone acetylation in the amygdala, and withdrawal of ethanol
triggers deacetylation in the same region (Pandey et al 2008). This deacetylation corresponds
with anxiety-type behaviors on the part of the mice, and the administration of TSA, a drug to
prevent the deacetylation, prevents the anxiety from manifesting. The researchers suggest that a
similar drug could be used in humans to affect the symptoms of withdrawal.
However, more information about the effects of alcohol on the human brain is necessary
before this concept can applied to medicine. Specifically, evidence that chronic alcohol
consumption affects chromatin structure in the amygdala of humans is currently lacking.
However, a novel method for detecting DNA methylation in the genome of human brain cells
has recently been developed (Johansson 2007). Using tissue obtained from a brain bank, global
DNA methylation has been analyzed in the prefrontal and motor cortex of chronic alcoholics.
The same method can be applied to the amygdala to discover whether the changes in histone
structure in mice are reflected in human chronic alcoholics and those going through withdrawal.
If these changes are conserved, it would provide evidence that drugs that inhibit deacetylation
may control human withdrawal symptoms as well as those in mice.
Methods
Tissue from the amygdala of thirty brains will be obtained from the New South Wales
Tissue Resource Center. Tissues will be selected from males over 18 years of age at the time of
death, with no other history of drug use or developmental or psychological disorders, and using
the selection criteria established in Johansson (2007). Fifteen of the brains will be from chronic
alcoholics with an average consumption of more than 80g of alcohol per day, and fifteen will be
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age-matched controls, with an average alcohol consumption of less than 10g of alcohol per day.
Of the brains from alcoholics, five will be from those whose alcohol habits continued unchanged
at the point of death, five will be from those who were actively suffering from withdrawal, and
five will be from those who were not suffering from the symptoms of withdrawal, according to
the next-of-kin being contacted for permission to use the tissues.
DNA methylation in each tissue sample will be analyzed by the LUMA method, featuring
quantified digestion of the DNA and reaction with lucifren to give off visible light captured by a
camera, as outlined in Johansson (2007). The results will be magnified by high throughput real
time PCR and organized by the application geNORM, which normalizes the expression ratio,
and analyzed with the Statistica software package, also as explained in Johansson (2007).
Discussion
This study aims to show that chromatin remodeling occurs in the amygdala during
chronic alcohol consumption and during withdrawal. The genome should be unusually
demethylated in chronic alcoholics as compared with controls, and those suffering from
withdrawal symptoms should display an abnormally high level of methylation, compared with
non-withdrawal alcoholics and with controls. Those who had ceased chronic alcohol
consumption and were not suffering from withdrawal symptoms should have levels of
methylation about equal with the controls.
If this hypothesis is correct, it agrees with the research in mice that alcoholism and
withdrawal affect chromatin remodeling in the amygdala. It provides further evidence for the
possibility of using drugs that affect chromatin remodeling to reduce the effects of withdrawal.
This has already been successful in mice, and if the same mechanism causes human withdrawal
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symptoms, it would be an extremely valuable treatment mechanism. It also supports the theory
that some of the effects of addiction are the result of chromatin remodeling in the brain.
Additional proof for this theory could help scientists focus their efforts in research attempts to
deal with addiction. It would be one more step in the process of finding solutions to the
problems of addiction that are widespread in modern society.
If the results do not show changes in methylation between alcoholics and alcoholics
suffering from withdrawal, it could be due to a number of factors. The effects of methylation
and acetylation may not be as directly linked in this area as has been assumed. A method for
directly testing acetylation in the human brain may be necessary to show this relationship. It also
may be that the relationship between acetylation in the amygdala and alcoholism in humans is
not the same as in mice. Mice are imperfect models of psychiatric disorders like addiction; it is
difficult to say that a mouse is truly addicted or suffering withdrawal. Because of the major
differences in humans, results from mice may not be replicatable in humans. This could be
evidence that animal models are not very helpful for studying complex human disorders. If the
results from mice do not match those of humans, they are not useful in attempting to understand
the human practice of addiction. However, given the usefulness of animal models in the field, it
seems more likely that using dead tissue instead of live mice or testing methylation instead of
acetylation would be the cause of any discrepancies.
The results would be inconclusive if the tissues showed wide variation in levels of
methylation that did not correlate to whether they were alcoholics or in withdrawal. This would
indicate that the natural variation in human chromatin structure is broader than the variation due
to alcohol. It would argue that there is not one pathway that alcohol addiction follows; not all
addicts have chromatin remodeling in the amygdala due to alcohol. This would not necessarily
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disagree with the mouse experiment; the mice were much more similar genetically and
environmentally than humans in the study, so differences due to one variable would be much
more visible. Researchers would have to look for way to isolate the results of alcoholism in
humans. If it could not be done, it may be that chromatin remodeling in the amygdala is not
enough to explain the symptoms of alcoholism and withdrawal. It may be one factor, but the
problem may be too complex to understand based on this single avenue of research.
This study only examined general methylation in the amygdala. Future research could
focus on specific genes affected by the chromatin remodeling process due to alcoholism. It
would also be useful to investigate how the methylation of the amygdala might interact with
methylation of the prefrontal and motor cortex. This methylation has already been shown to
correlate with alcoholism in humans; studying its effects in mice might also explain the effects of
alcohol addiction. Next-of-kin reports on alcoholism and withdrawal levels of deceased
individuals are not the most reliable measure of the symptoms of withdrawal. Future research
might involve tracking individuals involved in alcoholism studies who donate their brains to
science and doing this post-mortem research with knowledge of the individual symptoms.
However, this study alone should indicate whether the chromatin remodeling associated with
alcohol addiction and withdrawal symptoms in mice is also found in humans, which may
indicate possible avenues of therapy for those suffering from withdrawal.
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