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Epigenetic Jamming of GAD1 Promoter May Contribute to Schizophrenia

21 November 2007. On your favorite digital device, a broken switch can lead to frustration; in the case of schizophrenia, it may derail a life. According to a study in the October 17 Journal of Neuroscience, epigenetic blocking of the on/off switch, or promoter, for a gene that encodes an enzyme needed to make the neurotransmitter GABA may contribute to schizophrenia. Schahram Akbarian, of the University of Massachusetts Medical School in Worcester, and colleagues report that methylation of histone proteins in the chromatin helps control the amount of glutamate decarboxylase (GAD) that is expressed in human brain tissue during normal development and aging, and offer evidence that this regulation is perturbed in the disease, specifically in females. The researchers also report that clozapine might exert some of its effects via this dynamic mechanism for controlling gene expression.

Several lines of research link GABA (gamma-aminobutyric acid) abnormalities to schizophrenia (see related SRF news story), and particularly reduced levels of GAD67 (encoded by the gene GAD1), which facilitates breakdown of glutamate to generate GABA. In a 2006 literature review ( Akbarian and Huang, 2006), Akbarian and Hsien-Sung Huang, also at the University of Massachusetts, write, “Evidence … strongly suggests that altered GAD67 transcription is at the core of the molecular pathology of schizophrenia and related disorders, and therefore further in-depth analysis of GAD67-related transcriptional mechanisms bears promise to provide important insight into the neurobiology of psychosis.” In their new research—actually a series of studies—they take on that challenge.

Epigenetic processes enable cells with the same DNA sequence to develop differently, without directly altering the DNA. Since, in theory, they can be reversed, they beguile with the promise of new targets for drug development (see, e.g., Petronis, 2004 and related SRF news story). Among several different epigenetic processes that can alter transcription rates, the direct methylation of DNA has attracted the most attention in the context of psychiatric disease, particularly from the research team led by Erminio Costa and Alessandro Guidotti at the University of Illinois at Chicago (Costa et al., 2006).

In their current research Akbarian, first author Huang, and colleagues focused instead on histone methylation. Histones are protein "spools" around which DNA coils; together with other scaffolding proteins, they comprise chromatin. The addition of methyl groups to histones can affect the gene transcription machinery’s ability to access and read genes.

Akbarian and colleagues began by using normal postmortem brain tissue from 55 humans to study the prefrontal cortex as it develops from before birth to old age. They found that levels of mRNA associated with GAD1, and to a lesser extent GAD2, which encodes for GAD65 (another GABA-synthesizing enzyme), rise as the fetus develops and increases until around puberty. After that, GAD1 and GAD2 mRNA levels plateau or dwindle slightly.

With these changes come increased methylation at histone H3-lysine 4 (H3K4) at GAD1 and other GABA-related sites (GAD2, NPY [neuropeptide Y], and SST [somatostatin]). In particular, the addition of three methyl groups to form H3K4me3 reflects the occurrence of transcription. The study suggests that levels of H3K4me3 at the GAD1 promoter, as well as at other GABA-related gene sites, increase severalfold from before birth to childhood, and from childhood to adulthood.

Noting these changes, the researchers wondered what controls histone methylation at the GAD1 site. Of the various transcripts that encode enzymes with H3K4 methyltransferase activity, they found only one—mixed-lineage leukemia 1, or MLL1—that was expressed abundantly in the adult human prefrontal cortex and the mouse cerebral cortex. Furthermore, mice heterozygous for a truncated Mll1 allele had less H3K4me3 at GABA-related promoters.

According to Akbarian and associates, “It is remarkable that, according to the present study, chromatin structures in prefrontal cortex are subjected to progressive changes from prenatal to peripubertal stages. In addition, this process continues at some gene loci, including GAD1, throughout adulthood into old age.“ They think that understanding these developmental changes might open new doors to understanding what goes wrong in schizophrenia.

Perturbations in schizophrenia?
To investigate further, the researchers studied patterns of histone methylation and mRNA levels in postmortem tissue from 36 humans with schizophrenia and in matched controls. For GAD2, cases and controls looked similar. In contrast, females (but not males) with schizophrenia showed deficits in GAD1 mRNA and GAD1 H3K4me3 levels. The researchers also report a statistical association between these markers and age of onset: reduced GAD1 mRNA and GAD1 H3K4me3 levels were correlated with later age of onset.

Finally, the researchers linked declines in prefrontal GAD H3K4me3 and in GAD mRNA levels in patients to a haplotype at the 5’ end of the GAD1 site at a locus previously reported to be a risk factor for schizophrenia. They write, “We conclude that genetic polymorphisms around the proximal GAD1 promoter play an important role for chromatin alterations and transcriptional dysregulation in schizophrenia subjects.”

Studies suggest that antipsychotic drugs increase rodents’ Gad1 expression in the cerebral cortex, and Akbarian and associates thought they might do so via chromatin remodeling. To test this notion, they injected mice with either clozapine or haloperidol for 21 days. Clozapine, an atypical antipsychotic, seems to improve cognitive symptoms of schizophrenia more than the conventional antipsychotic haloperidol does, and it works better at getting neurons in the prefrontal cortex to fire in sync as they should (see SRF related news story).

Clozapine, but not haloperidol, tripled Gad1-associated H3K4me3 in the cerebral cortex of mice. It also ramped up Mll1 mRNA expression. The researchers then compared prefrontal cortex from nine humans with schizophrenia who had undergone clozapine treatment before death with tissue from matched controls who had received conventional antipsychotics. In clozapine-treated subjects only, H3K4me3 doubled at the GAD1 site. “Together, the animal studies and the human data support the notion that clozapine positively regulates MLL-1 mediated histone methylation at the GAD1 locus,” Akbarian and associates write. Notably, however, the prefrontal cortex of the clozapine-treated human subjects did not show a significant increase in GAD1 mRNA. The researchers discuss this finding as an important limitation of the study.

The challenge for future research will be to build upon the findings in this paper regarding chromatin remodeling at GABA-related gene sites under normal development and aging, and to help determine whether the preliminary findings vis-à-vis schizophrenia and antipsychotic drugs are supported.—Victoria L. Wilcox and Hakon Heimer.

Huang H-S, Matevossian A, Whittle C, Kim SY, Schumacher A, Baker SP, Akbarian S. Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters. J Neurosci. 2007 Oct 17; 27(42):11254-11262. Abstract

Comments on News and Primary Papers

Primary Papers: Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.

Comment by:  Dennis GraysonErminio Costa
Submitted 14 November 2007
Posted 14 November 2007

Comment by Dennis R. Grayson, Erminio Costa, and Alessandro Guidotti
Schizophrenia is a devastating disorder with a population-wide morbidity approaching 1 percent. The genetics of this disease are perhaps the most studied facet of the disorder, but the results of multiple linkage analyses across the entire genome have provided only limited insight into the underlying etiological factors that hallmark the disease. While linkage studies are informative, problems associated with the interpretation of complex genetics make their interpretation difficult. In fact, the inconsistencies associated with the results of recent meta-analyses of genome-wide scans and large sib-pair studies indicate that there is no replicable support for any of the currently considered candidate genes (Crow, 2007). Recent thinking suggests that the origins of the disease may lie in DNA sequence variations coupled with epigenetic dysfunction as a key etiopathogenic factor.

Several studies point to a defect in GABAergic transmission as a key factor underlying the schizophrenia phenotype. Over 10 years ago, a decrease in GAD67 (GAD1) expression and an increase in selected GABA-A receptor subunits in the frontal cortex of patients with schizophrenia was reported (Akbarian et al., 1995). We replicated and extended this finding to include reelin and GAD1 in frontal cortex (Impagnatiello et al., 1998; Guidotti et al., 2000), while others have since reported comparable findings in the hippocampus (Heckers et al., 2002). Numerous laboratories have proposed that the reduced expression of reelin, GAD1, and other mRNAs results in a decrease in interneuron inhibitory tone that has been described in schizophrenia patients (reviewed in Benes and Berretta, 2001; Guidotti et al., 2005; Lewis et al., 2005). The decreased inhibitory tone may cause a downregulation in post-synaptic dendritic spine protein synthesis and a disruption of the synchronized high-frequency pyramidal neuron firing rates that characterize changes in cortical function.

The most common mechanism proposed to account for the epigenetic changes observed in GABAergic neurons of schizophrenia patients is CpG methylation (Costa et al., 2002; 2007). Several labs have examined the methylation patterns of reelin in postmortem human material (Abdolmaleky et al., 2005; Grayson et al., 2005). More recently, global methylation patterns have been shown to increase in the cortex of humans across the lifespan (Siegmund et al., 2007). In their current paper, Huang et al., 2007 discuss another potentially important means of modifying promoter function. That is, alterations in chromatin remodeling mediated by changes in histone methyltransferases in GABAergic neurons are likely to affect the accessibility of selected promoters to the transcriptional machinery. What makes the current study so interesting is that the investigators have found changes in multiple promoters expressed in these neurons including those of GAD1, neuropeptide Y, and somatostatin. Interestingly, the reduced levels of GAD1 mRNA correlate with increased GAD1 promoter H3K4 trimethylation. The histone methyltransferase responsible is mixed-lineage leukemia 1 (MLL1). Mice heterozygous for the Mll1 locus show reduced GAD1 H3K4 trimethylation at GABAergic gene promoters. In addition, GAD1 H3K4 trimethylation and Mll1 occupancy are increased following treatment with the atypical antipsychotic clozapine. It seems clear from these and other studies that the epigenetic origins of schizophrenia are becoming increasingly evident and that they operate on multiple levels. It remains to be seen how DNA methylation and histone methylation cooperate in this cascade and why the defect is localized to GABAergic neurons. Do these mechanisms act in concert, or do they act independently? It seems reasonable that DNA methylation and histone methylation may converge, effecting a similar end result.


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

Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney WE, Jones EG. Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 1995 Apr 1;52(4):258-66. Abstract

Benes FM, Berretta S. GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder. Neuropsychopharmacology. 2001 Jul 1;25(1):1-27. Abstract

Costa E, Chen Y, Davis J, Dong E, Noh JS, Tremolizzo L, Veldic M, Grayson DR, Guidotti A. REELIN and schizophrenia: a disease at the interface of the genome and the epigenome. Mol Interv. 2002 Feb 1;2(1):47-57. Abstract

Costa E, Dong E, Grayson DR, Guidotti A, Ruzicka W, Veldic M. Reviewing the role of DNA (cytosine-5) methyltransferase overexpression in the cortical GABAergic dysfunction associated with psychosis vulnerability. Epigenetics. 2007 Jan-Mar ;2(1):29-36. Abstract

Crow TJ. How and why genetic linkage has not solved the problem of psychosis: review and hypothesis. Am J Psychiatry. 2007 Jan 1;164(1):13-21. 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

Guidotti A, Auta J, Davis JM, Di-Giorgi-Gerevini V, Dwivedi Y, Grayson DR, Impagnatiello F, Pandey G, Pesold C, Sharma R, Uzunov D, Costa E, DiGiorgi Gerevini V. Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder: a postmortem brain study. Arch Gen Psychiatry. 2000 Nov 1;57(11):1061-9. Abstract

Guidotti A, Auta J, Davis JM, Dong E, Grayson DR, Veldic M, Zhang X, Costa E. GABAergic dysfunction in schizophrenia: new treatment strategies on the horizon. Psychopharmacology (Berl). 2005 Jul 1;180(2):191-205. Abstract

Heckers S, Stone D, Walsh J, Shick J, Koul P, Benes FM. Differential hippocampal expression of glutamic acid decarboxylase 65 and 67 messenger RNA in bipolar disorder and schizophrenia. Arch Gen Psychiatry. 2002 Jun 1;59(6):521-9. Abstract

Impagnatiello F, Guidotti AR, Pesold C, Dwivedi Y, Caruncho H, Pisu MG, Uzunov DP, Smalheiser NR, Davis JM, Pandey GN, Pappas GD, Tueting P, Sharma RP, Costa E. A decrease of reelin expression as a putative vulnerability factor in schizophrenia. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15718-23. Abstract

Lewis DA, Hashimoto T, Volk DW. Cortical inhibitory neurons and schizophrenia. Nat Rev Neurosci. 2005 Apr 1;6(4):312-24. Abstract

Siegmund KD, Connor CM, Campan M, Long TI, Weisenberger DJ, Biniszkiewicz D, Jaenisch R, Laird PW, Akbarian S. DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. PLoS ONE. 2007 Jan 1;2(9):e895. Abstract

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Comments on Related News

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

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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.

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