Variability of DNA Methylation within Schizophrenia Risk Loci across Subregions of Human Hippocampus

Identification of 108 genomic regions significantly associated with schizophrenia risk by the Psychiatric Genomics Consortium was a milestone for the field, and much work is now focused on determining the mechanism of risk associated with each locus. Within these regions, we investigated variability of DNA methylation, a low-level cellular phenotype closely linked to genotype, in two highly similar cellular populations sampled from the human hippocampus, to draw inferences about the elaboration of genotype to phenotype within these loci enriched for schizophrenia risk. DNA methylation was assessed with the Illumina HumanMethylation450 BeadArray in tissue laser-microdissected from the stratum oriens of subfield CA1 or CA2/3, regions having unique connectivity with intrinsic and extrinsic fiber systems within the hippocampus. Samples consisted of postmortem human hippocampus tissue from eight schizophrenia patients, eight bipolar disorder patients, and eight healthy control subjects. Within these genomic regions, we observed far greater difference in methylation patterns between circuit locations within subjects than in a single subregion between subjects across diagnostic groups, demonstrating the complexity of genotype to phenotype elaboration across the diverse circuitry of the human brain

Induction of the GABA Cell Phenotype: An In Vitro Model for Studying Neurodevelopmental Disorders

Recent studies of the hippocampus have suggested that a network of genes is associated with the regulation of the GAD67 (GAD1) expression and may play a role in γ-amino butyric acid (GABA) dysfunction in schizophrenia (SZ) and bipolar disorder (BD). To obtain a more detailed understanding of how GAD67 regulation may result in GABAergic dysfunction, we have developed an in vitro model in which GABA cells are differentiated from the hippocampal precursor cell line, HiB5. Growth factors, such as PDGF, and BDNF, regulate the GABA phenotype by inducing the expression of GAD67 and stimulating the growth of cellular processes, many with growth cones that form appositions with the cell bodies and processes of other GAD67-positive cells. These changes are associated with increased expression of acetylated tubulin, microtubule-associated protein 2 (MAP2) and the post-synaptic density protein 95 (PSD95). The addition of BDNF, together with PDGF, increases the levels of mRNA and protein for GAD67, as well as the high affinity GABA uptake protein, GAT1. These changes are associated with increased concentrations of GABA in the cytoplasm of “differentiated” HiB5 neurons. In the presence of Ca2+ and K+, newly synthesized GABA is released extracellularly. When the HiB5 cells appear to be fully differentiated, they also express GAD65, parvalbumin and calbindin, and GluR subtypes as well as HDAC1, DAXX, PAX5, Runx2, associated with GAD67 regulation. Overall, these results suggest that the HiB5 cells can differentiate into functionally mature GABA neurons in the presence of gene products that are associated with GAD67 regulation in the adult hippocampus

Co-localization of GAD₆₇ with (A) GluR5 and (B) expression glutamate receptor subunits GluR6/7 in differentiated cells.

<p>Specific antibodies were used to detect glutamate receptor subunits. (<b>A</b>) GluR5 expression in differentiated HiB5 cells; (<b>B</b>) GluR6/7 expression in differentiated HiB5 cells. GluRs (red) was co-localized with GAD₆₇ (green).</p

Site-specific regulation of cell cycle and DNA repair in post-mitotic GABA cells in schizophrenic versus bipolars

GABA cell dysfunction in both schizophrenia (SZ) and bipolar disorder (BD) involves decreased GAD67 expression, although this change involves fundamentally different networks of genes in the 2 disorders. One gene that is common to these 2 networks is cyclin D2, a key component of cell cycle regulation that shows increased expression in SZ, but decreased expression in BD. Because of the importance of cell cycle regulation in maintaining functional differentiation and DNA repair, the current study has examined the genes involved in the G1 and G2 checkpoints to generate new hypotheses regarding the regulation of the GABA cell phenotype in the hippocampus of SZ and BD. The results have demonstrated significant changes in cell cycle regulation in both SZ and BD and these changes include the transcriptional complex (TC) that controls the expression of E2F/DP-1 target genes critical for progression to G2/M. The methyl-CpG binding domain protein (MBD4) that is pivotal for DNA repair, is significantly up-regulated in the stratum oriens (SO) of CA3/2 and CA1 in SZs and BDs. However, other genes associated with the TC, and the G1 and G2 checkpoints, show complex changes in expression in the SO of CA3/2 and CA1 of both SZs and BDS. Overall, the patterns of expression observed have suggested that the regulation of functional differentiation and/or genomic integrity of hippocampal GABA cells varies according to diagnosis and their location within the trisynaptic pathway

Synthesis of GABA and stimulation-induced release from differentiated HiB5 cells.

<p>(<b>A</b>) GABA was detected using a specific antibody against GABA. (<b>B</b>) GABA release was detected in artificial cerebrospinal fluid (aCSF) without KCl (basal) or with 100 mM KCl (stimulation) in the presence of Ca²⁺. GABA concentrations in aCSF were determined using HPLC. The data represent the mean ± SEM for GABA release from 6 independent culture experiments (***≤0.0004 vs baseline levels in differentiated cells).</p

Treatment scheme for chronic influence of growth factors on expression of GABA cell marker.

<p>Cells were maintained for two days under proliferation conditions (undifferentiated (undiff) as shown in the left picture). For the first 4 days of differentiation, a routine differentiation protocol for HiB5 was followed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033352#s2" target="_blank">methods</a> section): after two days at 39°C in N2 medium, PDGF (30 ng/ml) was added and the cells incubated for another 2 days. To study the influence of growth factors they were added at day 4 and cells incubated for another 2 days.</p

Expression pattern of mature neuron markers acetylated tubulin, MAP2 and PSD-95 and stem cell marker nestin in undifferentiated and differentiated HiB5 cells.

<p>Specific markers were detected using primary antibodies and immunofluorescence detection with DAPI as nuclear counter stain. Detectable levels of IF were associated with, acetylated tubulin (<b>A</b>); MAP2 (<b>B</b>); PSD-95 in differentiated HiB5 cells (<b>C</b>), while nestin (<b>D</b>) is only seen in undifferentiated cells.</p

Quantification of cells expressing differentiation stage-specific markers.

<p> <b>The data represent 100 cells/coverslips that were analyzed using cells grown on 5 different coverslips per marker and differentiation state. Statistical significance was calculated by one-way ANOVA, followed by Tukey test.</b></p>*<p>: <b>p<0.001 for GABA undiff. vs GABA diff., tubulin undiff. vs. tubulin diff., nestin undiff. vs. nestin diff., respectively Data are expressed as means ± SD.</b></p

Induction of expression of key GABA cell markers GAD₆₇ and GAT-1 in differentiated HiB5 cells.

<p>(<b>A, B</b>): Cells were differentiated at 39°C in N2 for two days, followed by addition of PDGF (30 ng/ml) for an additional two days. This was followed by stimulation for two days with growth factors. Quantitative (q) RT-PCR for GAD₆₇ or GAT1 was used to measure changes in mRNA expression in the presence N2 supplement plus PDGF alone; additional PDGF (P); or a combination of additional PDGF and BDNF (B; 50 and 100 ng/ml respectively). Values are expressed as fold increases in differentiated cells compared with undifferentiated controls (<b>GAD₆₇</b> vs undiff: *≤0.05; <b>GAT1</b>: vs undiff ***≤0.001). <b>C, D</b>: Western blot with β-actin as loading control was used for quantification of proteins. (*≤0.05 vs undiff). GAD₆₇ and GAT-1 were detected using specific antibodies. (<b>E</b>): DAPI (blue) was used as nuclear counter stain for GAD₆₇ containing cells (green). (<b>F</b>): GAD₆₇ (green) was co-localized with GAT1 (red) in the same differentiated cells (yellow overlay color) (<b>G</b>) GAD₆₇ (green) in undifferentiated HiB5 cells; DAPI (blue): cell nuclei.</p