23 February 2012. As the knotty search for candidate genes in schizophrenia continues, new information about how gene expression is controlled, and how these processes may be altered in the illness, is beginning to accumulate. The molecular mechanisms underlying altered gene expression in schizophrenia may include epigenetics, mitotically heritable changes that control gene activity without altering DNA sequence, as well as post-transcriptional changes mediated by microRNAs (miRNAs) (Gavin and Akbarian, 2011; see also SRF Current Hypothesis by Dennis Grayson).
Two new studies offer clues to the potential role of these processes in schizophrenia. One study, headed by Joel Kleinman and Barbara Lipska, both of the National Institute of Mental Health in Bethesda, Maryland, examined epigenetic changes in the dorsolateral prefrontal cortex (DLPFC) across the lifespan of healthy individuals, finding dramatic changes early in life. Alterations in this process might contribute to the postulated neurodevelopmental hypothesis of schizophrenia. A second study, also in the DLPFC, led by Claes Wahlestedt of Florida’s University of Miami, found altered expression of a particular microRNA, a short noncoding RNA sequence that causes repression of target mRNAs, in subjects with schizophrenia.
DNA methylation across the lifespan
In Kleinman and Lipska’s study, published online February 1 in the American Journal of Human Genetics, first author Shusuke Numata and colleagues investigated the role of epigenetics in the development of the human DLPFC. They focused on the epigenetic mechanism of DNA methylation, the transfer of a methyl group to a cytosine residue at the dinucleotide sequence CpG (adjacent cytosine and guanine nucleotides on the same DNA strand connected by a phosphodiester bond), typically associated with gene repression. The researchers scanned approximately 27,000 CpG sites in the 5’ promoter regions of genes, using tissue from 108 subjects ranging in age from 14 weeks prenatal to 84 years.
Numata and colleagues observed a distinct pattern of DNA methylation across the lifespan of healthy individuals. The fastest changes in DNA methylation were found prenatally, with levels changing by nearly 80 percent per year. Childhood methylation levels changed around two orders of magnitude slower, and post-childhood levels even slower still (approximately three orders of magnitude slower than the prenatal period). Interestingly, the transition from fetal age to childhood was associated with a reversal in the direction of DNA methylation, with over half of the CpG sites that overlapped between the two time points displaying this pattern. In general, demethylation predominated prenatally, while methylation levels climbed postnatally.
Kleinman's and Lipska’s groups have recently analyzed the DLPFC transcriptome in a larger developmental cohort that includes the subjects utilized in the current study (Colantuoni et al., 2011; see SRF related news story). Similar to the alterations in DNA methylation in the present study, they found that the largest changes in gene expression occurred during the transition from fetal to postnatal life. Coupled with the observed inverse correlations between DNA methylation and gene expression levels, these data provide strong evidence that DNA methylation changes during this time window correspond to a regulation of gene expression.
The researchers found prominent changes in DNA methylation during the switch from fetal to postnatal life in a number of genes such as DLG4, DRD2, NOS1, and NRXN1 that have previously been implicated in schizophrenia (Cheng et al., 2010; Dubertret et al. 2004; Cui et al., 2010; Yue et al., 2007). Since increased levels of the DNA methyltransferase DNMT1 have been found in schizophrenia (Veldic et al., 2005), the authors suggest that a rise in methyltransferase activity that alters the normal developmental trajectory may be a potential mechanism for altered DNA methylation in the illness.
MiRNA-132 and its targets in schizophrenia
Shifting gears to the regulation of gene expression that occurs post-transcriptionally, the Wahlestedt study, appearing online February 6 in the Proceedings of the National Academy of Sciences, examined the expression patterns of 854 miRNAs in DLPFC tissue from healthy subjects (n = 34) and those with schizophrenia (n = 35) and bipolar disorder (n = 31), obtained from the Stanley Medical Research Institute.
Through repression of their target mRNAs, miRNAs are known to be key regulators of a variety of biological functions, from neuronal migration during development to adult neurogenesis, and are thought to affect as many as 60 percent of protein-coding RNAs (Bartel, 2009). Recently, evidence for a role for these mini-repressors in schizophrenia has been growing (Perkins et al., 2007; Beveridge and Cairns, 2011).
After performing a false-discovery rate correction for multiple comparisons, first author Brooke Miller and colleagues observed a significant downregulation of one miRNA—miR-132—in schizophrenia subjects. MiR-132 was also significantly decreased in the same cohort using qPCR, and in a second cohort of 15 controls and 16 schizophrenia subjects from the Harvard Brain Bank. Of the 263 protein-coding targets of miR-132 identified using the TargetScan prediction database, 26 were upregulated in the Stanley cohort of schizophrenia subjects (including GATA2, PDE7B, and P250GAP), suggesting that miR-132 can have widespread effects on gene expression.
Interestingly, although 10 miRNAs were significantly dysregulated in subjects with bipolar disorder, miR-132 was not on the list. However, using a less stringent criterion for significance in the Stanley cohort, 10 miRNAs, including miR-132, were found to be altered, and changed in the same direction, across both diagnoses, consistent with recent data suggesting shared susceptibility between the two illnesses (see SRF related news story; SRF news story).
To explore the potential mechanisms underlying altered miR-132 in schizophrenia, the researchers examined its developmental trajectory in mouse PFC, finding that levels rose fourfold during postnatal weeks 2 through 4 (corresponding to adolescence in humans). Consistent with these findings, protein-coding gene targets of miR-132 (including DNMT3A and DPYLS3) were downregulated during this same time period.
In line with the NMDA hypofunction hypothesis of schizophrenia (see SRF Current Hypothesis) and previous work demonstrating that miR-132 can be regulated by NMDA receptors (Cheng et al., 2007), Miller and colleagues found that treating mice with the NMDA antagonist MK-801 during early postnatal development, or chronically in adulthood, produced downregulations of miR-132 expression and upregulation of its targets in the PFC. However, NR1 hypomorphic mice did not display reduced miR-132 expression, and thus the role of NMDA function on miR-132 requires further investigation.
Both DNA methylation and miR-132 exhibit large changes during development, and the two studies provide potential mechanisms for altered gene expression in schizophrenia. Of note, one of the miR-132 targets downregulated during development, DNMT3A, encodes for a DNA methyltransferase (Feng et al., 2010), suggesting a possible link between epigenetic- and miRNA-mediated gene regulation. Future studies are needed to explore this potential interaction, and to directly link both of these mechanisms to gene changes in schizophrenia.—Allison A. Curley.
Miller BH, Zeier Z, Xi L, Lanz TA, Deng S, Strathmann J, Willoughby D, Kenny PJ, Elsworth JD, Lawrence MS, Roth RH, Edbauer D, Kleiman RJ, Wahlestedt C. MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function. Proc Natl Acad Sci U S A. 2012 Feb 6. Abstract
Numata S, Ye T, Hyde TM, Guitart-Navarro X, Tao R, Wininger M, Colantuoni C, Weinberger DR, Kleinman JE, Lipska BK. DNA Methylation Signatures in Development and Aging of the Human Prefrontal Cortex. Am J Hum Genet. 2012 Feb 1. Abstract