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