Parallel changes in gene expression in peripheral blood mononuclear cells and the brain after maternal separation in the mouse

Background The functional integration of the neuro-, endocrine- and immune-systems suggests that the transcriptome of white blood cells may reflect neuropsychiatric states, and be used as a non-invasive diagnostic indicator. We used a mouse maternal separation model, a paradigm of early adversity, to test the hypothesis that transcriptional changes in peripheral blood mononuclear cells (PBMCs) are paralleled by specific gene expression changes in prefrontal cortex (PFC), hippocampus (Hic) and hypothalamus (Hyp). Furthermore, we evaluated whether gene expression profiles of PBMCs could be used to predict the separation status of individual animals. Findings Microarray gene expression profiles of all three brain regions provided substantial evidence of stress-related neural differences between maternally separated and control animals. For example, changes in expression of genes involved in the glutamatergic and GABAergic systems were identified in the PFC and Hic, supporting a stress-related hyperglutamatergic state within the separated group. The expression of 50 genes selected from the PBMC microarray data provided sufficient information to predict treatment classes with 95% accuracy. Importantly, stress-related transcriptome differences in PBMC populations were paralleled by stress-related gene expression changes in CNS target tissues. Conclusion These results confirm that the transcriptional profiles of peripheral immune tissues occur in parallel to changes in the brain and contain sufficient information for the efficient diagnostic prediction of stress-related neural states in mice. Future studies will need to evaluate the relevance of the predictor set of 50 genes within clinical settings, specifically within a context of stress-related disorders.


Background
The application of microarray techniques has provided insights into the multi-dimensional molecular nature of complex neuropsychiatric disorders. Studies have highlighted the value of using peripheral tissue targets [1,2], an approach based on the functional integration of neural-, endocrine-and immune-systems [3]. Regulatory exchanges between components of these systems provide a foundation for using peripheral tissue targets as indicators of neuropsychiatric states.
One of the earliest demonstrations that gene expression changes in peripheral blood mononucleoctyes (PBMCs) reflected disease states in the brain, was based on a rat model, where acute neural assaults resulted in gene expression changes in PBMCs within 24 hours [4]. Recent studies have focused on human neuropsychiatric disorders with more subtle disruptions in neurophysiology. Segman et al [1] were able to predict the onset and progression of post-traumatic stress disorder (PTSD), in recently traumatised patients. Similarly, Tsuang et al [2] showed that the microarray analysis of peripheral blood samples discriminated between patients clinically diagnosed with schizophrenia or bipolar disorder and healthy controls. Nevertheless, it remains to be established whether gene expression changes in peripheral tissue targets are paralleled by specific transcriptional alterations in neural tissues [1].
We have used the model of maternal separation, which is known to induce long term alterations in neurophysiology and stress-related behaviours in adult rodents [5,6] to investigate i) whether parallel changes occur in gene expression in three brain regions (the prefrontal cortex, hippocampus, and hypothalamus) and PBMCs and ii) whether gene expression changes in PBMCs could be used to predict the animal treatment group.

Animals and treatment
Maternal separation was carried out on C57BL/6 mice as previously described [6] with some modifications. Briefly, MS litters were separated from dams for 3 h a day, starting at 12 h 00 and ending at 15 h 00, from postnatal day (PND) 1 to 14. SH animals underwent brief daily handling. All subsequent procedures were carried out using males only, as the consequences of separation are gender specific [6].

Acute restraint stress, sacrifice, blood collection and brain dissections
Mice (N MS = 30, N SH = 30) were subjected to 10 min of acute restraint stress and allowed to recover for 20 min prior to sacrifice. Restraint stress was chosen as a means of acutely activating the Hypothalamic-Pituitary-Adrenal (HPA) axis (HPAA), which allowed for an assessment of possible differences in plasma corticosterone profiles (van Heerden et al, submitted manuscript). All mice were sacrificed, by means of cervical dislocation, immediately followed by decapitation and collection of trunk blood. Neural tissues: the (1) prefrontal cortex (PFC), (2) hippocampus (Hic) and (3) hypothalamus (HYP) were immediately dissected and submerged in RNALater ® (Qiagen Inc., USA).

Microarray processing and data analysis
Fifty-five samples, 15× PFC (8× MS and 7× SH), 10× Hic and 10× Hyp (5× MS and 5× SH, each and 20× PBMC (10× MS and 10× SH) were used for microarray processing, with a two-colour common reference design. Samples were matched, so that 10 individuals (5× MS and 5× SH) were completely represented in all tissues. A common reference pool was constructed by combining equal amounts (0.75 μg) of PFC and Hic RNA from both groups. Commercial pre-spotted, full mouse genome, microarray slides (OpArray™) were sourced from Operon (Operon Biotechnologies, Germany). Full details of RNA labelling, microarray hybridization, image capture and microarray data processing are given in Additional file 1: Supplementary Methods. Microarray data are available in the ArrayExpress database http://www.ebi.ac.uk/arrayexpress under accession number E-MEXP-2101.
Data normalization was done in R, using the Limma package [7]. Pre-processing and removal of batch effects were done using GEPAS http://www.gepas.org and ASCA-genes [8] respectively. Differentially expressed genes were identified using a concordance strategy [9], based on overlap between three statistically divergent approaches. Genes that had a P-value < 0.05, using both the Info statistic, from the ScoreGenes software package http:// www.cs.huji.ac.il/labs/compbio/scoregenes/, and the Tusher et al [10] Significance Analysis of Microarrays (SAM) implementation in the T-Rex module of GEPAS http://www.gepas.org, in addition to an absolute foldchange > 1.2 (where fold change is defined as the fold difference between MS and SH), were considered to be differentially expressed (DE).
All data clustering was done in the Tigr MultiExperiment Viewer V4.1 (TMEV, http://www.tm4.org) using a Pearson correlation metric with average linkage. Functional enrichment of GO terms within differentially expressed gene sets was evaluated using Blast2GO [11]. Gene set enrichment analysis on lists ordered according to SAM statistics was done using FatiScan http://www.babe lomics.org [12]. The PFC and Hyp lists were evaluated using 50 partitions, the PBMC list using 55 partitions and the Hic list using 60 partitions.
The efficiency of PBMC gene expression profiles at predicting the treatment class of samples (i.e. MS or SH) was evaluated with the Prophet module in GEPAS http:// www.gepas.org [13] using both the K-nearest neighbour (KNN) and Support Vector machine (SVM) algorithm options. Leave-one-out cross validation was used to counter selection bias whilst simultaneously assessing prediction efficacy.

Results and Discussion
Microarray data comparing the response of control and MS adult mice to stress was used to investigate the presence of a functional link between gene expression changes in the brain and PBMCs. In the first instance data was analysed to characterise the transcriptional response of three brain regions, the prefrontal cortex, the hippocampus and hypothalamus to stress, and to investigate whether a coordinated change in glutamatergic and GABAergic systems occurred in MS mice. Corresponding differences in gene expression in PBMCs of MS mice compared to control mice were also identified. Importantly, these differences could be used to predict the treatment status of mice.

MS produced gene expression differences in all tissues
Differentially expressed (DE) genes were identified in all tissues ( Figure 1A-D). A summary of all DE genes is provided in [see Additional file 2 Table S2], [see Additional  file 3 Table S3], [see Additional file 4 Table S4], and [see Additional file 5 Table S5]. The unsupervised hierarchical sample clustering of differentially expressed genes, produced clear group (MS or SH) separations within all tissues ( Figure 1E-H). No single gene was differentially expressed across all tissues.

Gene set enrichment analysis revealed significant functional themes
The FatiScan analysis revealed the significant enrichment of functional terms, in all tissues ( Figure 2 and Figure 3). In PBMC samples ( Figure 3B . These results suggest a functional shift in the immune system in PBMCs in MS mice, characterised by the coordinated down-regulation of energy requiring processes, such as protein synthesis and transport. This functional shift might reflect the well characterised mobilisation of energy and inhibition of further storage in response to stress [14].

Response of the glutamergic and GABergic systems in neural tissues after stress
DE genes and enriched functional terms from the PFC datasets highlighted the importance of the glutamatergic and GABAergic systems in the stress-related response of the MS mice. These two neurotransmitter systems constitute the major stimulatory (glutamate) and inhibitory (GABA) mechanisms of neurotransmission, and work counteractively to ensure optimal neuronal activity after stress [15]. Glutamatergic signalling was enhanced in MS mice possibly as a consequence of deficiencies in GABAergic mediated inhibitory mechanisms.
DE genes whose products are involved in the modulation of glutamatergic and GABAergic signalling included P2yr4 and Npvf (Figure 4). The activation of P2yr4 positively regulates glutamate release [16], whereas Npvf is an important inhibitor of GABAergic neurotransmission [17]. The over-expression of both these genes in the MS PFC tissue, points to a hyperactive glutamatergic system. Supporting this observation is the under-expression of Myo6 in the MS samples. Myo6 is crucial for the efficient endocytosis of postsynaptic glutamate receptors, with deficiencies resulting in increased excitatory neurotransmission [18]. Htr3a was also under-expressed in MS samples. This receptor is strongly associated with GABAergic neurons and interneurons which activate the GABA mediated inhibitory neurotransmission in the prefrontal cortex [19]. The coordinated under-expression of both pre-and post-synaptic component GO terms further supports the hypothesis of a hyperglutamatergic state in the PFC of MS mice (Figure 4). Specifically, genes supporting depletion of postsynaptic components in MS mice included three GABA A receptors (GABA A alpha-1 and -3, and GABA A gamma-3) ( Figure 4); such receptors mediate inhibition of neurotransmission with disruptions resulting in enhanced anxiety [20]. Genes supporting functional depletion of presynaptic components included two metabotropic glutamate receptors, mGluR3 and mGluR7 (Figure 4). These receptors participate in negative feedback mechanisms that inhibit presynaptic glutamate release. Results from the hippocampal gene expression dataset extend these observations, with the over-representation, in MS samples, of genes involved in ionotropic glutamate signalling ( Figure 4). Although this hyperglutamatergic theme was not readily apparent in either the DE genes or func-Differential gene expression results These findings are consistent with the central role of glutamate in the stress-response, in structures such as PFC and hippocampus. Stressors such as acute restraint have been shown to produce dramatic and rapid increases in glutamate levels primarily in the PFC, which ultimately culminates in HPAA activation and glucocorticoid secretion. In addition, the hippocampus is a major site of stress-associated glutamate action. The mechanisms which regulate glutamate action and release within this region function downstream of prefrontal cortical processes, constituting a secondary stress-response phase, which, unlike the PFC, is sensitive to neuroendocrine modulation [22]. The glutamatergic signature found here in both the PFC and hippocampus is therefore consistent with previous work.

Functional significance of gene expression changes in PBMC tissues
A large number of genes (418) were found to be differentially expressed between MS and SH individuals and included several genes whose products are important modulators of immune system function. Examples include Foxp3, an essential modulator of T cell function [23]; IL-17ra, the receptor target for the IL-17 mediated inflammatory pathway [24]; and Ccl5 (also known as Rantes), which regulates the activity of several cellular populations within the immune system [25].
The evidence obtained from the neural transcriptomes (combined with corticosterone and behavioural profiles; van Heerden et al Submitted Manuscript) indicates that pre-weaning treatment (MS or SH) result in differential stress-related profiles. Given this context, the gene expression information derived from the PBMC samples was evaluated in terms of its ability to derive accurate predictions of pre-weaning status of individuals.

PBMC gene expression profiles accurately predict sample classes
The classification and prediction of sample classes (MS or SH) using PBMC gene expression values, were found to be highly efficient. Using KNN (with 4 neighbours), 50 genes ( Figure 5; Table 1) were sufficient to accurately identify sample classes 19 out of 20 times. Most of the genes included in the predictor were over-expressed ( Figure 5B). SVM, however, only achieved this success rate using a minimum of 125 genes (with linear and radial kernels). Importantly, this 125 gene set consisted of the 50 genes included in Table 1, in addition to 75 other genes, which were the same for both algorithms (data not shown).
Of the 50 genes included in the predictor, 46 were functionally annotated. Of particular interest was the identification of 3 genes, Oxt, Cck and Adcy8 (all over-expressed), whose products are known to be important mediators of stress-and anxiety-associated behaviours (Table 1) [26][27][28]. Both Oxt and Cck are neuroactive hormones with previously described endogenous immunomodulatory properties [29,30]. These results confirm that the transcriptional profiles of peripheral immune tissues do indeed contain sufficient information for the efficient diagnostic prediction of stress-related neural states in mice. Products of these genes may participate in pathways that are particularly sensitive to stress-induced regulation of the immune system.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JHvH carried out the animal studies, microarray experiments, data analysis and drafted the manuscript. NI designed and supervised the study, and assisted with the writing of the manuscript. DM and AC assisted with the analysis of the microarray data. DJS and VR contributed to the conception and design of the study, and assisted in the editing of the final versions of the manuscript. All the authors read and approved the final manuscript.
Schematic summary of neural gene expression results in sup-port of a stress-related hyperglutamatergic state in MS brain samples Figure 4 Schematic summary of neural gene expression results in support of a stress-related hyperglutamatergic state in MS brain samples. Such a hyperglutamaterigc state could potentially result in elevated stress-induced corticosterone responses. Red indicates over-expression and blue under-expression, in MS samples, respectively. An asterisk indicates genes or functional classes that were found to be regulated in a coordinated manner. Glu = Glutamate, (+) indicates increased signalling activity, (-) indicates decreased signalling/acitivity.  Sample classification and prediction results