Infragranular gene expression disturbances in the prefrontal cortex in schizophrenia: signature of altered neural development?

Abstract

The development of the human neocortex gives rise to a complex cytoarchitecture, grouping together cells with similar structure, connectivity and function. As a result, the six neocortical laminae show distinct molecular content. In schizophrenia, many anatomical and neurochemical changes appear to be restricted to a subset of lamina and/or cell types. In this study, we hypothesized that supragranular (SG; laminae II-III) and infragranular layers (IG; laminae V-VI) of area 46 in the human prefrontal cortex will show distinct and specific transcriptome alterations between subjects with schizophrenia and matched controls. To enhance sample homogeneity, we compared the gene expression patterns of the SG and IG layers of 8 matched middle-aged male subjects with schizophrenia to 8 pairwise matched controls using two replicate DNA microarrays for each sample. The study revealed strong disease-related laminar expression differences between the SG and IG layers. Expression changes were dominated by an overall underexpression of the IG-enriched genes in the schizophrenia subjects compared to normal control subjects. Furthermore, using a diagnosis-blind, unsupervised clustering of the control-derived SG or IG-enriched transcripts, the IG-enriched markers segregated the subjects with schizophrenia from the matched controls with a high degree of confidence. Importantly, multiple members of the semaphorin gene family reported altered gene expression, suggesting that the IG gene expression disturbances in subjects with schizophrenia may be a result of altered cortical development and disrupted brain connectivity.

Figure 1. Experimental design

All PFC BA 46 samples were harvested using laser dissection microscopy (LCM). For each of the 16 postmortem subjects (8 SCZ and 8 matched CNT) 4 samples were obtained, 2 from supragranular (SG: laminae II-III) and 2 from the underlying infragranular (IG: laminae V-VI) cortical layers. Each sample was amplified by a single-round isothermal linear amplification and hybridized to a HG_U133plusV2 Affymetrix oligonucleotide microarray. This generated a dataset of 64 arrays, which were subjected to gcRMA normalization and several distinct statistical analyses. In subsequent analyses SG-IG enriched genes were overlaid with the diagnosis comparison, identifying genes SG-IG laminar markers that also showed differential expression in the brain of subjects with schizophrenia.

Figure 2. SG-enriched genes show no expression change in subjects with schizophrenia

A. ALR_SCZ-CNT distribution of the 158 SG-enriched transcripts. Each symbol denotes a single gene, X axis corresponds to the ALR_SCZ-CNT in the SG samples, Y axis shows the mean intensity for each of the IG-enriched genes across all samples. Note that the transcript population ALRs are comparably distributed across the two sides of the unity line, indicating no SG-enriched gene overexpression or underexpression in SCZ samples. B. The dendrogram is derived from GSEA two-way hierarchical clustering of expression levels from the 158 SG-enriched transcripts. The SCZ (red bar) and CNT samples (green bar) could not be separated based on the expression levels of the SG-enriched genes.

Figure 3. IG-enriched genes show a robust expression repression in subjects with schizophrenia

A. ALR_SCZ-CNT distribution of the 226 IG-enriched transcripts. Each symbol denotes a single gene, X axis corresponds to the ALR_SCZ-CNT in the IG samples, Y axis shows the mean intensity for each of the IG-enriched genes across all samples. Note the robust shift of the transcript population to the left (underexpression in SCZ). B. The dendrogram is derived from GSEA two-way hierarchical clustering of expression levels from the 226 IG-enriched transcripts. Although the disease status was not the entered in the clustering, IG markers perfectly clustered the SCZ (red bar) and CNT samples (green bar) in the IG samples.

Figure 4. Two-way hierarchical clustering of SG-IG enriched genes that showed differential expression in the SCZ-CNT comparison

The figure denotes a two-way clustering of the normalized expression levels for 51 SG-IG enriched genes altered in schizophrenia. In the vertical dendrogram, each arm represents a single sample (red-schizophrenia; green – control), rows denote gene probesets with NCBI accession numbers and gene symbols. Each pixel corresponds to a log₂-normalized expression level in a single sample. The intensity of red is proportional to transcript increase, while the blue intensity is proportional to transcript decrease. Based on the expression levels of these 61 probe sets, the vertical dendrogram perfectly separated out the schizophrenia (red) and control samples (green). Numeric data is represented in Table 2.

Figure 5. Gene expression profile across subjects for three Semaphorin family members

In each figure, sample pairs are denoted on X axis (AVG corresponds to mean), Y axis denotes RMA normalized log2 expression level. Blue symbols represent control subjects, red symbols correspond to subjects with schizophrenia. Note the downregulation of all three SEMA genes across the subject pairs.