Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors

Abstract

Cortical tubers in patients with tuberous sclerosis complex are associated with disabling neurological manifestations, including intractable epilepsy. While these malformations are believed to result from the effects of TSC1 or TSC2 gene mutations, the molecular mechanisms leading to tuber formation, as well as the onset of seizures, remain largely unknown. We used the Affymetrix Gene Chip platform to provide the first genome-wide investigation of gene expression in surgically resected tubers, compared with histological normal perituberal tissue from the same patients or autopsy control tissue. We identified 2501 differentially expressed genes in cortical tubers compared with autopsy controls. Expression of genes associated with cell adhesion, for example, VCAM1, integrins and CD44, or with the inflammatory response, including complement factors, serpinA3, CCL2 and several cytokines, was increased in cortical tubers, whereas genes related to synaptic transmission, for example, the glial glutamate transporter GLT-1, and voltage-gated channel activity, exhibited lower expression. Gene expression in perituberal cortex was distinct from autopsy control cortex suggesting that even in the absence of tissue pathology the transcriptome is altered in TSC. Changes in gene expression yield insights into new candidate genes that may contribute to tuber formation or seizure onset, representing new targets for potential therapeutic development.

Figure 1

Histopathological features of TSC—cortical tubers. A–B. Hematoxylin/Eosin (HE) staining; representative photomicrographs of cortical tubers (TSC) showing an area of cortical dislamination, containing different cell types, such as dysplastic neurons (arrows in B), reactive astrocytes (arrowheads in B) and giant cells (asterisks in B). C–E. NeuN staining showing the disorganization of the neuronal component within the cortical tuber (C) compared to perituberal (D) and control (E) cortical specimens. Scale bar in A: A, C–E: 250 µm; B, 27 µm.

Figure 2

Gene expression profiles. A. Hierarchical clustering on the set of genes (2501) that are significantly (and twofold) different between control and TSC specimens demonstrates a nice separation between the cortical tuber (CT1‐CT4) specimens and autopsy controls (AC). The dendrogram at the top displays the relationship of the samples based on their pattern of gene expression. Each column represents the gene expression profile on one specific array with each row representing the expression level of a specific gene. The expression levels are depicted in three colors; blue: low expression, white: medium expression and red: high expression; deeper shade indicates larger difference. B. Hierarchical clustering on the set of genes (111) that were significantly (and twofold) different between controls and perituberal tissue. The hierarchical clustering on the set of genes that were significantly (and twofold) different shows nice separation between the two groups, indicating that the samples are more similar within each condition. C. Scatter plot showing the correlation between average signal intensities in TSC and autopsy control samples. Data points represent average signal intensity (²log; n = 4 for each condition) calculated from Affymetrix signal intensities for all present genes expressed in the cortical tuber (TSC) and the autopsy control specimens. Red dots represent the significantly changed genes with at least a twofold change in gene expression and a P‐value < 0.01. Blue dots represent the genes that do not fulfill this criterion. D. Scatter diagram of the fold change in the cortical tuber specimens (compared to the autopsy controls) as a function of the P‐value. E. Scatter diagram of the fold change in the perituberal specimens (compared to the autopsy control specimens) as a function of P‐value. The pink dots represent the genes associated with cell adhesion or the immune/inflammatory response.

Figure 3

Validation of gene expression data with quantitative real‐time PCR. Increased expression levels of serpinA3, CCL2, CX3CR1, ECM2, VCAM1 and Integrin β4 in the cortical tubers were confirmed with quantitative real‐time PCR (A), while lower expression levels were observed for GAD67, GLT1, GABRA5 and Kir3.1 (B). Increased expression levels of genes associated with either the immune/inflammatory response or cell adhesion were confirmed in the perituberal regions compared to autopsy control specimens (C). Expression levels in cortical tuber specimens (n = 6) or perituberal specimens (n = 4) were compared to levels in autopsy control specimens (n = 7). Expression levels were corrected for the expression levels of TBP and normalized to control expression levels. The error bars represent SEM and * represents a P‐value < 0.05 (Student's t‐test).

Figure 4

CCL2, SerpinA3 and Integrin β1 immunoreactivity in cortical tubers (TSC). A–D. CCL2 immunoreactivity (IR). A. Moderate neuronal IR is observed in histologically normal cortex (CTX; insert), without detectable glial staining. B. Strong IR is observed in cortical tubers (TSC). CCL2 IR is detected in dysplastic neurons of different size and shape (arrows in C), in reactive glial cells (arrow‐heads in C) and in giant cells (arrows in D). E–F. SerpinA3 IR in CTX (E) and TSC (F). E. SerpinA3 IR is not detected in histological normal cortex and white matter (Wm, insert). F. Strong SerpinA3 IR is observed in the cortical tuber, with prominent expression in giant cells (arrows in F and insert a; and insert c, positive giant cell in Wm) as well as in reactive glial cells (insert b: arrow‐heads indicated reactive astrocytes surrounding a negative dysplastic neuron) and inflammatory cells (microglia/macrophages) surrounding blood vessels (arrow‐heads in insert a and insert a'). G–J. Integrin β1 IR in CTX (G) and TSC (H–J). G. Integrin β1 is only detected in endothelial cells in histological normal cortex (arrows and insert). H. Strong integrin β1 IR is detected in TSC; IR is mainly observed in giant cells (arrows in H–J), while dysplastic neurons have low or undetectable IR (arrow‐heads in H,I). Scale bar in J: A and G: 200 µm, B, E, F, H: 150 µm; C, I, J and inserts in F: 40 µm; D: 80 µm.