Schizophrenia Research Forum - A Catalyst for Creative Thinking

SfN 2005: Nature or Nurture—Epigenetics in Neuronal Responses

10 December 2005. Is it nature or nurture? Societies have mulled this over ever since the concept crept into our collective consciousness. Today, we know there is no simple answer and that human biology or behavior are rarely, if ever, explained by either phenomenon alone. Epigenetics, the study of changes to the genome that, unlike mutations, do not alter the DNA sequence, has exposed just how inextricably linked nature and nurture truly are. It is not surprising then, that the theme served as an epicurean delight at the 35th Annual Conference of the Society for Neuroscience in Washington, D.C., last month, in the form of a symposium of uniformly high quality.

Epigenetics is of interest to a growing number of biologists and clinicians. It can help to explain such disparate phenomena as twin discordance (see SRF related news story) and atypical social bonding (see Fries et al., 2005). At the molecular level, it likely plays crucial roles in neurotransmission (see, e.g., Stadler et al., 2005) and learning and memory in mammals (see SRF related news item). It reveals potential mechanisms underlying various central nervous system disorders ranging from schizophrenia (see Dong et al., 2005) to Rett syndrome (see SRF related news item), and it even suggests strategies for treatment of schizophrenia (Sharma, 2005). In fact, researchers are currently developing small molecules that perturb methylation and acetylation, two of the most consequential epigenetic changes, in the hope that they may be turned into drugs to treat a variety of diseases, including schizophrenia (see Tremolizzo et al., 2005).

To fully understand epigenetics, one must appreciate why it exists. At the Washington symposium, co-chairs Jonathan Pollock and Christine Colvis from the National Institute on Drug Abuse, Bethesda, Maryland, reminded the audience that epigenetics has its roots in adaptation. As organisms become more complex with longer generational times, it becomes harder and harder for them to adapt through natural selection, Pollock suggested. In short, where genetic change cannot keep pace with environmental change, complex organisms have evolved other means to adapt. Specialized tissues such as neurons have arisen to allow organisms to react rapidly to external influences. But that requires that specific subsets of genes be turned on and off in specialized cells. This is where chromatin remodeling enters the picture; epigenetic change, therefore, is a key element of adaptation based on genetic reprogramming.

It is easy to envisage how the apparatus for genetic reprogramming, once it had evolved to facilitate tissue differentiation in multicellular organisms, was co-opted for more immediate benefit, such as the response to environmental stimuli. Frances Champagne, University of Cambridge, England, reported on how something as simple and innocuous as maternal care can begin the process of chromatin remodeling right out of the womb. Work from Champagne’s and other laboratories reveals that rat pups who get licked and groomed the most turn out to do better in tests of learning and memory, such as the Morris water maze. They also have higher levels of both N-methyl-D-aspartate (NMDA) glutamate receptors and synaptophysin, a synaptic marker, in their brain, suggesting molecular adaptation. Well-groomed pups have higher levels of brain glucocorticoid receptor and appear less anxious than pups that get licked less. Notably, long-time Champagne collaborator Michael Meaney and colleagues at Douglas Hospital Research Center, Montreal, Canada, confirmed in 1999 that this pup behavior is epigenetic rather than inherited. If pups born to so-called “low licking/grooming” mothers are instead reared by “high licking/grooming” mothers, they will behave just like their adopted siblings (Francis et al., 1999). Meaney's collaborator in Montreal, Alain Gratton has extended this work to one of the most popular endophenotype models in schizophrenia research—prepulse inhibition, which may underlie sensorimotor gating deficits in the disorder (see SRF related news item). Gratton's work indicates that changes in maternal care affect mesocorticolimbic dopamine systems, presumably by epigenetic mechanisms, leading to deficits in sensorimotor gating (Zhang et al., 2005)

Champagne and colleagues have since probed epigenetic changes that underlie behavioral differences in rat pups. She found, for example, that the promoter region of the glucocorticoid receptor (GR) has 17 potential methylation sites and that pups born to mothers that lick less have much higher levels of GR promoter methylation, particularly at one specific methylation site, number 16. These methylation differences are not apparent at birth, but gradually emerge after 5 to 6 days of postnatal care. It turns out that these methylation patterns have molecular consequences. They prevent the binding of transcription factors, such as nerve growth factor-inducible protein A (NGFI-A), Champagne revealed.

These epigenetic changes need not be permanent; this is important when one considers epigenetics in terms of disease pathology or drug addiction. Champagne reported that when rats born to low licking/grooming mothers are given the histone deacetylase (HDAC) inhibitor trichostatin A, then by adulthood their methylation pattern changes to that of rats brought up by high licking/grooming mothers.

Acetylation of histones and methylation of DNA are almost as intertwined as the strands of nucleic acids themselves. Champagne showed that hypermethylation of the GR promoter correlates with hypoacetylation of histone H3. Because the HDAC inhibitor promotes acetylation of the H3 histone, demethylation of the GR promoter ensues, followed by increased expression (see Weaver et al., 2004). Indeed, Champagne showed that infusion of trichostatin A into adult rats leads to greater binding of NGFI-A to the GR promoter. She also revealed that directly stimulating methylation by administering methionine, a precursor to the methyl donor S-adenosyl-methionine, can achieve a similar reversal of maternal programming. Some of this work just appeared in the November 23 Journal of Neuroscience (see Weaver et al., 2005).

Epigenetics and Drug Abuse
In a related vein, Arvind Kumar, from the University of Texas Southwestern Medical Center, Dallas, reported how chronic and acute exposure to drugs of abuse can cause epigenetic changes around a variety of promoters. The long-term effects of drug addiction have been explored for some years. It has become apparent that they can lead to changes in gene expression in the brain, particularly in the striatum, where many of the neurons take part in reward pathways that can lead to addiction. But exactly how those changes occur remains mysterious.

Kumar’s work centers on epigenetic changes in rodent striatum in response to either chronic or acute cocaine administration. While acute cocaine induces expression of the transcription factor cFos, chronic exposure to the drug desensitizes cFos levels back to normal and instead leads to the accumulation of a truncated form of FosB (δFosB). Using chromatin immunoprecipitation experiments, or ChIP, Kumar probes the relation between cocaine and covalent modifications—phosphorylation, acetylation, and phosphoacetylation—of histones on target promoter regions in rats.

He reported that acute administration of cocaine leads to a fivefold increase in phosphoacetylation of histone H3 on the cFos promoter, but no change in newly acetylated histone H3. Histone H4 acetylation does increase about threefold, however. Chronic administration of the drug, on the other hand, leads to increased acetylation of histone H3 around the FosB promoter, Kumar reported, but no changes in histone H4. The ChIPs failed to detect any changes in histone acetylation patterns on promoters that do not respond to the drug, such as those for β-tubulin or tyrosine hydroxylase, suggesting that the phosphorylation and acetylation changes are specific to cocaine-induced promoters.

Kumar has also looked at changes occurring around other gene promoters. Expression of the protein kinase Cdk5 (see Alzheimer Research Forum SfN 2005 report), for example, is unaffected by acute cocaine but is induced by chronic exposure. Would the chromatin pattern mimic those seen for FosB? Kumar found that not only was acetylated histone H3 increased on the Cdk5 promoter, but that δFosB also bound to it under chronic cocaine administration, suggesting that δFosB may play a direct role in cocaine addiction, a possibility that has been hotly debated.

And like Champagne, Kumar and colleagues have modified the addiction pattern using deacetylase inhibitors. Kumar reported that both trichostatin A and sodium butyrate, a competitive inhibitor of HDACs, enhanced histone H3 phosphoacetylation in response to the drug when administered to rats, and that the animals scored much higher on a test that measures the reward component of their response to cocaine. Overexpression of histone deacetylase, on the other hand, dramatically decreased scores in this behavioral test, confirming the importance of histone acetylation in cocaine addiction. Most of this work was just published in the October Neuron (see Kumar et al., 2005). Kumar and colleagues are now using “ChIP on chip” assays, probing chromatin immunoprecipitation samples with DNA promoter chips, or microarrays, in a genome-wide search for promoters that are hyper- and hypoacetylated after chronic cocaine exposure. So far, nearly 85 promoters have been isolated that have increased acetylated H3 and H4 histones following chronic cocaine administration, and 48 promoters where acetylation of H3 and H4 decreased, he reported.

Epigenetics and Methylation
Richard Goodman, Oregon Health Sciences University, Portland, reported his ChIP experiments to find genes that bind to CREB, or cyclic AMP response element (CRE) binding protein. Though it has been known for many years that CREB binds to CRE, one of the quintessential promoter elements, there are still aspects of CRE/CREB biology that are poorly understood. As Goodman pointed out, in PC 12 cells there is a CRE sequence in the promoter for the somatostatin gene, yet neither CREB nor CREB binding protein (CBP) bind to it. This goes to show that CRE binding is not constitutive, Goodman suggested. Instead, it may depend on how the DNA is programmed in individual cells.

Goodman and colleagues use a sophisticated screening method called SACO, or serial amplification of chromosome occupancy, designed to be unbiased, to identify regions of DNA that bind CREB. SACO combines a ChIP experiment with serial analysis of gene expression, or SAGE (see SRF related news story), to identify DNA in chromatin segments pulled down by CREB antibodies. Using this approach, the researchers have identified thousands of potential genes regulated by CREB (see Impey et al., 2004).

In his SfN presentation, Goodman focused on one of these regions, which happens to harbor a non-coding sequence. In fact, about 20 percent of the sequences identified by the SACO experiment did not code for protein, said Goodman. One of the identified sequences lies upstream of a microRNA (miRNA) sequence called miR132. This microRNA is expressed in neurons and its CRE element does bind to CREB. In fact, expression of miR132 increases dramatically when cells are treated with brain-derived growth factor—the response is even greater than that seen for cFos.

So what does miR132 do? Goodman reported that the microRNA strongly induces neurite outgrowth and that antisense miR132 has the opposite effect. By using a prediction algorithm (MIRANDA, see Enright et al., 2003), he and colleagues identified a putative binding site in the 3’untranslated region (UTR) of the gene encoding the GTPase-activating protein, p250GAP. Goodman reported that miR132 inhibits expression of p250GAP and that that, in turn, promotes neurite outgrowth. This is an example of a CREB-regulated miRNA that regulates growth of neurites by responding to extrinsic trophic cues. The work appeared last month in PNAS (see Vo et al., 2005).

As for epigenetics, miR132 and other miRNAs identified in the SACO screen may also bind to other 3’UTRs, hinted Goodman, most notably that for methyl-CpG binding protein 2 (MeCP2), which is mutated in Rett syndrome. Though MeCP2 was initially identified as a transcriptional repressor that binds to methylated DNA, researchers recently showed that it is involved in RNA splicing, as well (see SRF related news story). Because the MeCP2 gene has a very large 3’UTR that possibly binds up to three miRNAs identified in the SACO screen, CREB signaling may mediate RNA splicing and play an important role in the regulation of methylation-linked transcriptional repression.

Epigenetics in Long-Term Memory
Last but not least, Mark Mayford, Scripps Research Institute, La Jolla, California, also addressed CREB signaling in his studies of epigenetic mechanisms in memory formation. Mayford described experiments to address the role of nuclear calcium signaling in the CREB-mediated induction of protein synthesis that is required for long-term memory.

Using the inducible tetracycline promoter system, Mayford and colleagues generated mice that express calmodulin binding protein (CaMBP) only in the nucleus of neurons in forebrain—the system allows expression to be turned off by adding doxycycline to the diet. Mayford checked the system by inducing seizures in adult mice. In control animals, seizures lead to activation of CREB (by phosphorylation on serine 133) in the CA1 neurons of the hippocampus, but this is blocked in the transgenic animals.

In most electrophysiological tests, the CaMBP mice behave just as controls. Excitatory postsynaptic potentials and paired-pulse facilitations are normal, for example. But they do behave differently in learning and memory tests. When CaMBP is on, latency increases in the Morris water maze and the mice show no preference for the quadrant where the hidden platform is located. If the calmodulin inhibitor is off, then the mice do just as well as control animals. Fear conditioning and novel object recognition tests revealed that mice expressing CaMBP have normal short-term memory but impaired long-term memory, Mayford reported.

Epigenetics entered this line of study when Mayford asked what targets Ca2+/calmodulin might activate to facilitate long-term memory. One likely target, Mayford suggested, is CBP, or CREB binding protein. CBP has histone acetyltransferase (HAT) activity and HATs can activate transcription by unraveling the chromatin to expose promoter elements. They have also been implicated many times in learning and memory (see related Alzheimer Research Forum conference report; as well as SRF related news story). When Mayford and colleagues expressed a dominant-negative CBP, rendered HAT-less by a point mutation, then spatial memory was impaired. Long-term memory declines, while short-term memory is unaltered. Furthermore, trichostatin A, the deacetylase inhibitor used by Champagne and Kumar above, can rescue animals expressing the dominant-negative CBP, showing once again that histone acetylation and chromatin modification play a key role in behavioral response to the environment. For more detailed reading on the symposium, see a monograph prepared in advance by the presenters (Colvis et al., 2005).—Tom Fagan.

Comments on News and Primary Papers
Comment by:  Robert Fisher
Submitted 24 December 2005
Posted 3 January 2006

Dr. Eric Nestler at UT Southwestern Medical Center, Dallas, has long postulated addiction as a 50/50 percent split between genetic predisposition and biological changes as a result of substance abuse and the brain's accommodation to the same. I have found this particular hypothesis to be quite useful in treating addicts and alcoholics in inpatient and outpatient settings. They usually are able to easily grasp the concepts on a reasonably scientific level. This approach allows them to avoid guilt, shame, ect., that are typically strong predictors of success or failure in treatment. It is an even more successful tool in working with co-occurring Axis I disorders.

View all comments by Robert Fisher

Comments on Related News

Related News: New Genetic Variations Link Schizophrenia and Bipolar Disorder

Comment by:  Mary Reid
Submitted 28 September 2006
Posted 29 September 2006

It's of interest that Vazza and colleagues suggest that 15q26 is a new susceptibility locus for schizophrenia and bipolar disorder. I have suggested that reduced function of the anti-inflammatory SEPS1 (selenoprotein S) at 15q26.3 may reproduce the neuropathology seen in schizophrenia.

View all comments by Mary Reid

Related News: New Genetic Variations Link Schizophrenia and Bipolar Disorder

Comment by:  Patricia Estani
Submitted 5 October 2006
Posted 6 October 2006
  I recommend the Primary Papers

Related News: Epigenetic Analysis Finds Widespread DNA Methylation Changes in Psychosis

Comment by:  Dennis Grayson
Submitted 26 March 2008
Posted 27 March 2008
  I recommend the Primary Papers

The paper by Mill et al. is one of the first comprehensive attempts to examine changes in methylation across the entire genome in patients with various diagnoses of mental illness. The study is well designed, extensive, and uses fairly new technology to examine changes in methylation profiles across the genome. In the frontal cortex, the authors provide evidence for psychosis-associated differences in DNA methylation in numerous loci, including those involved in glutamatergic and GABAergic transmission, brain development, and other processes linked with disease etiology. Methylation in the frontal cortex of the BDNF gene is correlated with a non-synonymous SNP previously associated with major psychosis. These data provide further support for an epigenetic origin of major psychosis, as evidenced by DNA methylation-induced changes likely important to gene expression.

In many ways, this seems reminiscent of the trend in genetics several years ago when the inclination was to move from single gene loci association and linkage studies to genomewide scans. The only downside of the approach is that what one gains in information, one (at least initially) loses in biology. That is given the wealth of new findings uncovered; we now need to go back and examine these results in light of what we know regarding gene function in neurobiology and cognition. Of course, this is the trend, now that microarrays have increased our capacity to look at all things at the same time. The flipside is that it will take several large-scale studies of this sort to better understand which findings are replicable and which are not. That is, do the results of the Mill paper agree with data obtained and carried out by laboratories using the methyl DIP or MeCP2 ChIP assays coupled with microarrays. While these experiments ask different questions, the implication is that there may be some degree of overlap in comparing these different methodologies. While this may be premature, there is a sense that this information will be available shortly.

Finally, I would like to focus on recent findings regarding the methylation of the reelin promoter. These authors (Mill et al.) and Tochigi and colleagues (Tochigi et al., 2008) have found that the reelin promoter is not hypermethylated in patients with schizophrenia. In fact, Tochigi et al., 2008, found that the reelin promoter is not methylated at all using pyrosequencing. However, several groups (Grayson et al., 2005; Abdolmaleky et al., 2005; Tamura et al., 2007; Sato et al., 2006) have shown that the human reelin promoter is methylated in different circumstances. Interestingly, there is little consensus in the precise bases that are methylated in these latter studies. Our group (Grayson et al., 2005) performed bisulfite treatment of genomic DNA and sequencing of individual clones. Moreover, we analyzed two distinct patient populations. The clones were sequenced at a separate facility. What was intriguing was that the baseline methylation patterns in the two populations was different, and yet several sites stood out as being relevant in both. We mapped methylation to the somewhat rare CpNpG sites proximal to the promoter. Interestingly, these bases were located in a transcription factor-rich portion (Chen et al., 2007) of the promoter and in a region that shows 100 percent identity with the mouse promoter over a 45 bp stretch. We have also been able to show that changing one of these two bases to something other than cytosine reduces activity 50 percent in a transient transfection assay. So the question becomes, How do we reconcile these disparate findings regarding methylation? As suggested by Dr. McCaffrey, the answer may lie in regional differences that arise due to the nature of the material available for each study. We have found a degree of reproducibility by using human neuronal precursor (NT2) cells for many of our studies. At the same time, this cell line is somewhat artificial and cannot be used to reconcile differences found in human tissue. Perhaps it might be prudent to examine material taken by using laser capture microdissection to enrich in more homogenous populations of neurons/glia. In moving ahead, it might be best to now focus on the mechanism for these differences in methylation patterns and try to understand the biology associated with the new findings (Mill et al., 2008) as a starting point.


Abdolmaleky HM, Cheng KH, Russo A, Smith CL, Faraone SV, Wilcox M, Shafa R, Glatt SJ, Nguyen G, Ponte JF, Thiagalingam S, Tsuang MT. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. Am J Med Genet B Neuropsychiatr Genet. 2005 Apr 5;134(1):60-6. Abstract

Chen Y, Kundakovic M, Agis-Balboa RC, Pinna G, Grayson DR. Induction of the reelin promoter by retinoic acid is mediated by Sp1. J Neurochem. 2007 Oct 1;103(2):650-65. Abstract

Grayson DR, Jia X, Chen Y, Sharma RP, Mitchell CP, Guidotti A, Costa E. Reelin promoter hypermethylation in schizophrenia. Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9341-6. Abstract

Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, Jia P, Assadzadeh A, Flanagan J, Schumacher A, Wang SC, Petronis A. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Am J Hum Genet. 2008 Mar 1;82(3):696-711. Abstract

Sato N, Fukushima N, Chang R, Matsubayashi H, Goggins M. Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers. Gastroenterology. 2006 Feb 1;130(2):548-65. Abstract

Tamura Y, Kunugi H, Ohashi J, Hohjoh H. Epigenetic aberration of the human REELIN gene in psychiatric disorders. Mol Psychiatry. 2007 Jun 1;12(6):519, 593-600. Abstract

Tochigi M, Iwamoto K, Bundo M, Komori A, Sasaki T, Kato N, Kato T. Methylation status of the reelin promoter region in the brain of schizophrenic patients. Biol Psychiatry. 2008 Mar 1;63(5):530-3. Abstract

View all comments by Dennis Grayson

Related News: Epigenetic Forces May Blaze Divergent Heritable Paths From Same DNA

Comment by:  Shiva SinghRichard O'Reilly
Submitted 2 February 2009
Posted 3 February 2009

The methylation difference between twins is clearly demonstrated using newer methods in this publication. However, conceptually it’s an old story now. A quick PubMed search for "monozygotic twins and non-identical" yielded a total of 7,653 publications. There is no doubt that the more we look, the more difference we will find between monozygotic twins. Also, monozygotic twin differences in methylation and gene expression are expected to increase with age. It is also affected by a variety of genetic and environmental factors. We have come a long way in genetic research on twins and the time has come to modify our thinking about monozygotic twins as "non-identical but closest possible" rather than as "identical." They started from a single zygote, but have diverged during development and differentiation including upbringing.

The implication of the published results is that the methylation (epigenetic) differences (in monozygotic twins) will be powerful in any genetic analysis of disease(s). Once again, it is probably more problematic than usually assumed. Also, it is particularly problematic for behavioral/psychiatric disorders including schizophrenia. The reason is multi-fold and includes the effect of (known and unknown) environment including pregnancy, upbringing, drugs, life style, food, etc. All these are known to affect DNA methylation and gene expression. As a result, they add unavoidable confounding factors to the experimental design. It does not mean that epigenetics is not involved in these diseases. Rather, directly establishing a role for methylation in schizophrenia will be challenging. A special limitation is the fact that methylation is known to be cell-type specific, and perfectly matched affected and normal (twin) human brain (region) samples for necessary experiments are problematic and methylation studies on other cell types may or may not be informative.

View all comments by Shiva Singh
View all comments by Richard O'Reilly