Epigenetic mechanisms expressed in basal ganglia GABAergic neurons differentiate schizophrenia from bipolar disorder

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Abstract

In the cerebral prefrontal cortex (PFC), DNA-methyltransferase 1 (DNMT1), the enzyme that catalyzes the methylation of cytosine at carbon atoms in position 5 in CpG dinucleotides, is expressed selectively in GABAergic neurons and is upregulated in layers I and II of schizophrenia (SZ) and bipolar disorder patients with psychosis (BDP).

To replicate these earlier findings and to verify whether overexpression of DNMT1 and the consequent epigenetic decrease of reelin and glutamic acid decarboxylase (GAD) 67 mRNA expression also occur in GABAergic medium spiny neurons of the caudate nucleus (CN) and putamen (PT) of SZ and BDP, we studied the entire McLean 66 Cohort (Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA) including SZ and BDP, which were matched with nonpsychiatric subjects.

The data demonstrate that in GABAergic medium spiny neurons of CN and PT, unlike in GABAergic neurons of layer I and II PFC, the increased expression of DNMT1 and the decrease of reelin and GAD67 occur in SZ but not in BDP.

This suggests that different epigenetic mechanisms must exist in the pathogenesis underlying SZ and BDP and implies that these disorders might involve two separate entities that are characterized by a well-defined neuropathology.

Introduction

Current studies have consistently shown that the prefrontal cortex (PFC) GABAergic interneurons of psychotic patients [schizophrenia (SZ) and bipolar disorder with psychosis (BDP)] express a downregulation of glutamic acid decarboxylase (GAD)67 (one of the two decarboxylases that synthesize GABA) and reelin (an extracellular matrix protein that is preferentially synthesized and secreted by GABAergic interneurons) (Benes et al., 1992, Akbarian et al., 1995, Impagnatiello et al., 1998, Guidotti et al., 2000, Fatemi et al., 2000, Eastwood and Harrison, 2003, Woo et al., 2004, Lewis et al., 2005). Upon secretion into the extracellular matrix, reelin adheres to the dendritic shafts and surrounds dendritic spines of cortical pyramidal neurons. This protein, perhaps by impinging on synaptically located integrin receptors, modulates event-related protein synthesis and may influence dendritic spine expression density (Costa et al., 2001, Liu et al., 2001, Dong et al., 2003), markedly changing LTP and cognitive function expression (Larson et al., 2003, Carboni et al., 2004, Beffert et al., 2005, Qiu et al., 2006).

Reelin and GAD67 promoters are embedded in large CpG islands and express methylation consensuses (Grayson et al., 2005). In the PFC of SZ patients, a decrease of reelin expression was associated with cytosine hypermethylation in the promoter region of the gene encoding for this protein (Grayson et al., 2005, Abdolmaleky et al., 2005). The regulatory role played by promoter CpG island methylation in the expression level of reelin can be inferred by an increase by up to 80 fold occuring the human reelin promoter following hypomethylation (Chen et al., 2002).

In the PFC of SZ and BDP patients, we have also quantified the expression of DNA methyltransferase 1 (DNMT1), which catalyzes the methylation of the carbon atom in position 5 of cytosines in CpG dinucleotides of various gene promoter regions of GABAergic neurons. We found that in these PFC neurons, DNMT1 is highly expressed whereas this enzyme cannot be detected in pyramidal neurons (Veldic et al., 2004, Ruzicka et al., in press). Moreover, DNMT1 expression is increased in a subset of cortical GABAergic interneurons in SZ and BDP patients. For example, it is increased in cortical layers I, II, and IV GABAergic neurons but not in GABAergic neurons of layers III, V and VI (Veldic et al., 2005, Ruzicka et al., in press). In PFC GABAergic neurons of SZ and BDP, the extent of the DNMT1 increase was accompanied by a related decrease of reelin and GAD67 expression (Veldic et al., 2004, Veldic et al., 2005). Hence, these studies suggest that the downregulation of GAD67 and reelin, or that of other genes expressed in cortical GABAergic neurons of psychotic patients, may be mediated by a 5-cytosine hypermethylation of the promoter CpG dinucleotides elicited by the increased expression of DNMT1 (Grayson et al., 2006).

Postmortem studies of human brain suggest that SZ may be associated with a GABAergic neuron downregulation detected not only in the cortex but also in the striatum (Impagnatiello et al., 1998). High affinity binding studies with [3H]muscimol show an increase in the number of GABAA recognition sites in the caudate nucleus (CN) of SZ patients (Hanada et al., 1987). In addition the expression levels of reelin mRNA are decreased by more than 70% in the CN of SZ patients compared to matched nonpsychiatric subjects (NPS) (Impagnatiello et al., 1998). In previous studies, we have shown that CN GABAergic neurons of SZ patients overexpress DNMT1 whereas reelin expression is downregulated (Veldic et al., 2004).

The goal of the present study is to replicate in the McLean 66 Cohort (Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA) earlier findings of a DNMT1 mRNA increase in GABAergic neurons in Brodmann's area (BA) 9 and BA10 (Veldic et al., 2004, Veldic et al., 2005, Ruzicka et al., in press) and to verify whether there is an overexpression of DNMT1 and a parallel downregulation of reelin and GAD67 in CN and putamen (PT) medium spiny GABAergic neurons, in SZ and BDP patients.

Section snippets

Tissue collection

Tissue samples isolated from CN, PT, and BA9 of NPS, SZ or BDP patients were obtained from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA (Table 1). All specimens were fixed in 4% formaldehyde. RNA quality was established by the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA (http://www.brainbank.mclean.org/) (Table 2). The brain was cut rostro-caudally in 0.5-cm-thick slices along the coronal axis, starting from the frontal pole. We received Section

CN

Fig. 1 (a,b,c) shows that in the CN of the NPS group several but not all GAD65/67-containing neurons express detectable amounts of DNMT1 mRNA. In contrast, in the CN of SZ patients (Fig 1d,e,f ), almost every GAD65/67-positive neuron is DNMT1 mRNA-positive.

Three-dimensional counting established that the average number of DNMT1 mRNA-positive neurons is increased in the SZ group by ∼ 40% compared to the corresponding number of DNMT1-positive neurons in NPS (Fig. 2a). In contrast in subjects with

DNMT1 is overexpressed in GABAergic neurons of caudate/putamen from SZ but not from BDP patients

From the study of DNMT1 mRNA expression in the CN, PT, and BA9 of the entire McLean 66 cohort, it was found that in the telencephalon: a) DNMT1 was exclusively expressed in GABAergic neurons, b) in SZ patients, the number of GABAergic neurons expressing DNMT1 mRNA was increased not only in BA9 layers I, II, III–IV, but also in CN and PT, and that c) in BDP, the number of DNMT1 mRNA-positive neurons was increased in layers I and II of BA9, but not in GABAergic neurons of CN, PT, or layers III–IV

Acknowledgement

This work was supported in part by the National Institute of Mental Health Grants RO1MH71667 (to E.C.) and RO1MH70855 (to A.G.).

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