Monthly Archives: July 2018

31Jul/18

Epigenetics and the Treatment of Substance Misuse Disorders

Danielle Victoriano interned at Carrier Clinic this summer, where she worked on clinical trials. She learned under the direction of Andrew Walsh. During some down time, she labored on a series of research projects and articles. This piece on Epigenetics is the second one to be published.

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In recent years, the growing number of substance abusers in the United States has become a topic of national discussion. As the death toll from overdoses continues to skyrocket—with about 63,600 deaths in 2016, a number 21% higher than in 2015—substance abusers, family members, substance abuse counselors, and scientists are desperately searching for solutions that would help curb this national crisis. From all this turmoil has arisen a potential aid in the fight against substance abuse: epigenetics.

The topic of epigenetics cannot be breached without a better understanding of DNA, the material inside cells that serves as the language of life. DNA is the command center for why we look the way we look, and sometimes, why we behave the way we behave.

DNA or deoxyribose nucleic acid can be broken down to four bases: Adenine, Thymine, Cytosine and Guanine. Each of these bases are attached to a phosphate group and a sugar, and this combination of bases, sugars, and phosphate groups form the double-helix ladder that often comes to mind when we think about DNA.

Image result for dna

However, this viewpoint is wholly incomplete. It must be understood that the DNA ladder also actually wraps itself around proteins called histones.  The tightness from the wounding around these histone proteins regulate which parts of the gene are expressed and repressed. The closer the histone proteins are to each other, the tighter the DNA is wound, and thus, the harder it is for the DNA-encoding machinery to access the DNA material. The further away the histone proteins are to each other, the looser the DNA is wound, which means that it is easier for gene expression to occur.

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The distance by which histone proteins are from each other and the tightness of the DNA wounding around these proteins is determined by two chemical groups. Attachments of methyl groups on either the DNA itself or on the histone proteins are associated with repressing, or turning off the expression of a gene, while acetyl groups are associated with expression, or turning on the expression of a gene. Methylating the DNA and/or methylating the histone proteins tightens up the DNA-protein interaction, while acetylating the histone proteins opens up the gene and allows for expression.

To think about it more simply, imagine a flashlight that’s been turned on. Now, imagine a hand covering it. To open up the fingers over the flashlight means that light will pass through. To close the fingers over the flashlight means that light will not be able to pass through. This is similar to how epigenetic modifications to the human genome works. The light represents the fact that the DNA material will be there, it is whether or not the hand is open or closed that will factor in to if the gene is actually repressed or expressed. When the hand is open, there is expression, and when the hand is closed, there is repression.

Thus, epigenetics is the study of how the human genome is modified not through changes in the base pair sequence, but how the structure of DNA and protein is modified through attachments of chemical groups that alter the configuration.

The blossoming field of epigenetics has paved the way to discovery of epigenetic markers brought by substance abuse. By epigenetic markers, this means that genes have been identified to be methylated or acetylated differently from the norm.

One substance misuse disorder that is of high importance to public policy is the abuse of opiates. According to the Center for Disease Control, in 2016, around 66% of the 63,600 drug-overdose related deaths involved opioid use.

Researchers have found that in opioid addictive patients, there is more expression of a certain neuro-excitatory gene, GRA1, that would account for the addiction’s withdrawal symptoms such as restlessness, muscle twitching, and dilated pupils. This overexpression can be attributed to hyperacetylation of the neuro-excitatory gene. Hyperacetylation means that the gene is constantly being expressed, which leads to more expression than usual.

Alcohol is yet another substance misuse disorder that has been identified to have epigenetic markers. With alcohol use, researchers have found dysregulated amount of protein factors in the brain through abnormal methylation and acetylation patterns in some genes. In turn, during the active influence of alcohol, there is high concentration of the protein Arc, which is associated with decreased levels of anxiety and stress. This relationship results in alcohol-dependence to combat negative emotions that can be seen in many individuals with alcohol misuse disorders.

Knowing the existence of these epigenetic markers, and being able to identify and locate where they are in the human genome opens up a whole new realm of opportunity in terms of treatment options in substance abuse recovery. Potentially, the current rates of relapse that follow inpatient detoxification treatments could be curbed. As of 2015, it is reported that more than 85% of substance misuse patients relapse and return to substance use within a year following treatment. Since withdrawal symptoms are a trigger for relapse, imagine being able to control and limit those symptoms by targeting the epigenetic markers that caused the symptoms in the first place. Imagine being able to de-acetylate the neuro-excitatory gene, GRA1, that is constituently turned on in those with opioid abuse. This would most likely make early recovery easier, less painful, and less daunting.

Medications that are currently being examined to combat substance abuse are histone deacetylase inhibitors (HDAC inhibitors) and DNA methylation inhibitors (DNMT inhibitors).  These medications would attempt to re-regulate the imbalance that have been produced by epigenetic modifications in the body, by reversing or inhibiting the effects of methyl groups and acetyl groups.

Though research is still in its infancy, with no current research on how these medications affect humans, results have shown to be promising. One research study looked at the effects of having histone deacetylase inhibitors in rats with alcohol addiction and found that with HDAC medication, these rats are less likely to seek out or self-administer alcohol.

In terms of non-medication related treatments, there has been a promising finding that exercise can be used to reverse some of the epigenetic modifications made from chronic alcohol abuse. It has been found through rat studies that exercise has restored the brain physiology that has been altered by chronic drinking. Alcohol-addicted mice that were treated with regular exercise had improved memories and brain activities when compared to the non-treatment group.

Despite these success stories in controlled environments, there is still a lot more that needs to be done before this new technology can be used in humans. Currently, we simply do not have the technology to selectively choose genes that are specifically targeted by different addictive stimuli. However, we are hopeful that this new field will take off. With growing interest from research institutes in the national and global arenas, we can hope that the awaited future of epigenetics is not that far.

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Danielle Victoriano is a member of the Princeton University Class of 2019. She studies Ecology & Evolutionary Biology and plans on going to Medical School. She can be reached at dzv@princeton.edu

 

30Jul/18

Epigenetics

Daniel Shen interned at Carrier Clinic this summer, where he worked on clinical trials. He learned under the direction of Andrew Walsh. During some down time, he labored on a series of research projects and articles. This piece on Epigenetics is the first one to be published.

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The National Suicide Prevention Lifeline is a 24-hour suicide prevention network that takes 1.5 million calls per year nationally, or roughly 4,000 calls per day[1]. Following the tragic deaths of celebrities Kate Spade and Anthony Bourdain earlier this June, this number rose by 65% for several days, an increase of more than 2,500 calls per day[2]. Such spikes are typical following media coverage of celebrity suicides; similar statistics were reported after musician Chester Bennington’s death in 2017, and in the months following actor Robin Williams’s death in 2014, national suicide morbidity rates increased by nearly 10% according to a recent study from Columbia University[3].

Regardless of the precise psychological and sociological factors behind the so-called “celebrity-suicide effect”, clearly these high-profile incidents seem to touch a painful hidden nerve within our nation. According to the National Institute of Mental Health, one fifth of all Americans, or nearly 45 million individuals, are currently struggling with some form of mental illness[4]. When news of Ms. Spade and Mr. Bourdain’s deaths broke, conversation within the media focused particularly on an alarming CDC statistic reporting a 30% increase in the national suicide rate since 1999[5]. (For reference, the national population grew by only 16% since then[6].) Yet despite the prevalence and severity of these illnesses, nearly 60% of suffering adults do not seek treatment, and if the caller volume spikes following Ms. Spade and Mr. Bourdain’s deaths are any indicator, this statistic likely suffers from under-reporting[7]. The minority that do seek treatment are nearly always prescribed medications that often come with a litany of adverse side effects – nausea, drowsiness, weight gain, and muscle tremors, to name a few – that add further stress to an already difficult situation.

Epigenetics is a growing area of biological research which has rapidly gained the attention of medical researchers within the past two decades and is now being considered as a potential strategy for devising effective psychiatric treatments. Promising developments have already begun in other arenas; recently elucidated epigenetic mechanisms behind some cancers have already led to the creation of novel cancer drugs, and similar progress is being made for many other non-communicable diseases[8].

In basic terms, epigenetics can be defined as the study of the biological mechanisms behind gene expression – that is, how cells can switch certain segments of DNA on or off in order to perform specific functions. For instance, although your skin cells and brain cells all contain the same DNA and thereby the same genes, your skin cells do not sprout dendrites and your brain cells do not secrete oils/sweat because both cell types have switched off the gene segments encoding for inappropriate and unnecessary features. Proper gene expression is essential for an organism’s growth and survival, and many diseases are the result of switches being in the improper state: some gene is turned off when it should be on, or vice versa. For example, many cancers arise when genes encoding for crucial DNA repair/proofreading proteins become switched off, leaving cells more vulnerable to cancer-causing mutations. This leaves medical researchers with a tantalizing prospect: if the onset of a certain disease is due to improperly switching on/off certain genes, could treatment simply be a matter of reversing the switches back to normal?

Psychiatric epigenetics research is based on the hypothesis that psychiatric disorders such as major depressive disorder (MDD) and schizophrenia have epigenetic markers – that is, these diseases are the result of either overexpression or underexpression of certain genes within brain cells, resulting in altered brain activity and behavioral symptoms. Furthermore, these markers can be either hereditary or environmentally triggered, and by reversing the pathological gene switches back to normal, the disease can be mitigated. Encouraging findings from cancer research and other non-psychiatric disciplines have already shown this to be a promising hypothesis8. MDD, schizophrenia, and generalized anxiety disorder (GAD) rank among the most common mental disorders in America, and consequently have been of primary interest for epigeneticists searching for markers[9]. Due to the sheer complexity of the brain, progress has been slower in the psychiatric realm compared to non-psychiatric arenas, as evidenced by the relative dearth of literature on the topic. Nevertheless, studies from animal models and post-mortem tissue studies have already yielded encouraging results; a few of these studies are highlighted below.

Major depressive disorder

Several lines of evidence have implicated brain-derived neurotrophic factor (BDNF), a protein responsible for healthy neuron function, as a key factor in the onset of MDD, with decreased expression of BDNF in the hippocampus being associated with depressive symptoms[10]. In 2009, a team from the University of Alabama used rat models to investigate whether or not the gene encoding for BDNF experienced any epigenetic alterations in depressed individuals[11]. They did so by subjecting a group of rat pups to abusive conditions such as social deprivation and maternal maltreatment to induce depressive behavior, then comparing their brain cells with those of a control group. What they found was that in the depressed rats, the BDNF gene exhibited signs of increased DNA methylation, a chemical modification of DNA that acts as an off-switch for gene expression. Furthermore, experimental treatments with a DNA methylation inhibitor, designed to undo this chemical modification, yielded promising results. Crucially, this epigenetic marker appeared to be heritable between generations as well: when female individuals from the depressed rat group were allowed to mate, their offspring also exhibited the same methylation patterns and the same behavioral symptoms. This suggests that epigenetics may offer a sobering framework by which the long-known hereditary aspects of mental illness can be understood.

Schizophrenia

 γ-aminobutyric acid, or GABA, is a neurotransmitter responsible for reducing neuronal excitability in the brain, among other functions. It has been hypothesized that for individuals suffering from schizophrenia, the mechanisms driving GABA production are defective in certain neurons, resulting in increased neural activity and pathological behavioral symptoms[12]. In 2005, a team of Harvard researchers looked for markers at the RELN gene within GABA-producing neurons, hypothesizing that defects in RELN production are a major factor behind GABA deficiencies[13]. Comparing post-mortem brain tissue between schizophrenic and non-schizophrenic individuals, they indeed found evidence of DNA methylation – an epigenetic off-switch – at this gene within brain cells of the frontal lobe in the schizophrenic samples. Moreover, their results were consistent with a previous study examining RELN expression in patients with bipolar disorder[14]. Given the similarity of psychosis symptoms between these two illnesses, this link suggests that epigenetics may provide a window by which the mechanisms behind these diseases can be better understood, leading to more effective treatments for a multitude of conditions.

Anxiety disorders

The hypothalamic-pituitary-adrenal (HPA) axis is a complex system consisting of the hypothalamus, pituitary gland, and adrenal glands and can be thought of as the “stress-system” of the body, forming the link between brain activity and hormone production. Corticotropin-releasing hormone receptor 1 (CRHR1) is a crucial protein that contributes to activating our bodies’ stress response via this system. A recent 2016 study from the Weizmann Institute of Science in Israel used rat models to investigate whether or not the gene encoding for CRHR1 was affected in rats with anxiety disorders (induced through various means)[15]. They found that for anxious rats, the CRHR1 gene was under-methylated compared to healthy rats, suggesting that this protein was being overexpressed in anxious individuals and resulting in overactivation of the stress response. When they treated the anxious mice with an experimental drug designed to restore methylation, they found that many of their symptoms subsided, demonstrating that epigenetics can indeed be a promising avenue for designing new pharmacological therapies.

Next steps

The above studies all present a positive correlation between specific epigenetic signatures and the onset of psychiatric disorders, suggesting that epigenetics research is indeed a promising direction for future diagnosis and treatment methods for these disorders. Nevertheless, the field of psychiatric epigenetics is still very much in its infancy, as evidenced by the relative dearth of literature on the subject compared to other disease studies like cancer. Moreover, all of the drugs that have been developed thus far (including the cancer drugs), promising though they may be, have been pan-inhibitors – that is, they affect the entire genome rather than specifically targeting one gene, raising concerns about potentially dangerous side-effects that are not fully understood. Reaching the point where epigenetic treatments can act with specificity and accuracy on single genes requires a more extensive understanding of our cells’ epigenetic mechanisms in all of their complexity, which is beyond our current knowledge. Given that our cells regularly erase and re-write epigenetic markers on their own as part of normal functioning, elucidating these mechanisms is certainly possible, and the moment we gain the ability to selectively modify epigenetic markers on single genes will herald a paradigm shift in medicine and human history.

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Daniel S. Shen is a member of the Princeton University Class of 2019 and studies within the Department of Music. He is planning on going to Medical School. He can be reached at dsshen@princeton.edu

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[1] Draper, J. (2015, January 05). National Suicide Prevention Lifeline: The First Ten Years. Retrieved July 05, 2018, from https://www.sprc.org/news/national-suicide-prevention-lifeline-first-ten-years

[2] Ravitz, J. (2018, June 13). Calls to suicide prevention hotline spiked after celebrity deaths, but what’s the next step? CNN. Retrieved July 5, 2018, from https://www.cnn.com/2018/06/12/health/suicide-hotline-increase/index.html

[3] Fink DS, Santaella-Tenorio J, Keyes KM (2018). Increase in suicides the months after the death of Robin Williams in the US. PLoS ONE 13 (2): e0191405. https://doi.org/10.1371/journal. pone.0191405

[4] Mental Illness. (n.d.). Retrieved July 05, 2018, from https://www.nimh.nih.gov/health/statistics/mental-illness.shtml

[5] Stone DM, Simon TR, Fowler KA, et al (2015). Vital Signs: Trends in State Suicide Rates — United States, 1999–2016 and Circumstances Contributing to Suicide — 27 States. MMWR Morb Mortal Wkly Rep 2018;67:617–624. DOI: http://dx.doi.org/10.15585/mmwr.mm6722a1.

[6] Population data retrieved July 5, 2018, from https://data.worldbank.org.

[7] Substance Abuse and Mental Health Services Administration, Racial/Ethnic Differences in Mental Health Service Use among Adults. HHS Publication No. SMA-15-4906. Rockville, MD: Substance Abuse and Mental Health Services Administration, 2015. Retrieved July 5, 2018, from https://www.samhsa.gov/data/sites/default/files/MHServicesUseAmongAdults/MH

[8] Allis, C., & Jenuwein, T. (2016). The molecular hallmarks of epigenetic control. Nature Reviews Genetics. http://dx.doi.org/10.1038/nrg.2016.59

[9] U.S. DALYs Contributed by Mental and Behavioral Disorders. (n.d.). Retrieved July 6, 2018, from https://www.nimh.nih.gov/health/statistics/disability/us-dalys-contributed-by-mental-and-behavioral-disorders.shtml

[10] Castren E, Rantamaki T. The role of BDNF and its receptors in depression and antidepressant drug action: Reactivation of develop- mental plasticity. Dev Neurobiol 2010;70:289–97.

[11] Roth TL, Lubin FD, Funk AJ, Sweatt JD. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol Psychiatry 2009; 65:760 – 769.

[12] Costa E, Chen Y, Davis J, Dong E, Noh JS, Tremolizzo L et al. REELIN and schizophrenia: a disease at the interface of the genome and the epigenome. Mol Interv 2002;2:47–57.

[13] Abdolmaleky HM, Cheng KH, Russo A, Smith CL, Faraone SV, Wilcox M et al. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. Am J Med Genet B Neuropsychiatr Genet 2005;134B:60–6.

[14] Fatemi SH, Earle JA, McMenomy T. 2000. Reduction in Reelin immunor- eactivity in hippocampus of subjects with schizophrenia, bipolar disorder and major depression. Mol Psychiatry 5:654–663, 571.

[15] Evan Elliott, Sharon Manashirov, Raaya Zwang, Shosh Gil, Michael Tsoory, Yair Shemesh, Alon Chen. Dnmt3a in the Medial Prefrontal Cortex Regulates Anxiety-Like Behavior in Adult Mice. Journal of Neuroscience 20 January 2016, 36 (3) 730-740.