Abstracts

RATIONALE: Tuberous sclerosis (TSC) is an autosomal dominant disorder highly associated with epilepsy that results from mutations in one of two genes. Cortical tubers in TSC are developmental malformations of the cerebral cortex characterized by disorganized lamination, disrupted radial organization of neurons, and the presence of dysplastic neurons (DNs) and giant cells (GCs) that exhibit abnormal dendritic arborization and cytomegaly. We have previously demonstrated increased expression of the cell adhesion molecule ICAM-1 in tubers. We now investigate the expression of genes and proteins that modulate ICAM-1 expression and identify activation of a proinflammatory cytokine and cell death pathway in tubers.
The objective of these experiments is to define enhanced expression of cytokine and cell signaling proteins in tubers using cDNA array, Western, and immunohistochemical assays.
METHODS: Tuber specimens were obtained intraoperatively or at post-mortem exam from 15 TSC patients. Poly (A) mRNA was amplified from tuber sections and homogenates and used to probe cDNA arrays containing select candidate genes. Protein lysates from tubers were used for Western assay. Some tuber specimens were also fixed, paraffin embedded, and then sectioned for immunohistochemical analysis. The candidate genes and proteins analyzed included ICAM-1, tumor necrosis factor alpha (TNFa), nuclear factor kappa B (NFk- B), mitogen activated protein kinase (MAPK/Erk1, Erk2), Fas, Fas ligand (Fas-L), caspase 8, and the macrophage marker CD 68. For comparison, expression of these genes and proteins was determined in peri-tuberal cortex from TSC patients and control cortex obtained post-mortem.
RESULTS: We confirmed the robust expression of ICAM-1 mRNA and proteins in tubers. The expression of TNFa, NFkB, and MAPK was increased in tuber protein lysates by Western assay compared with control cortex or peri-tuberal cortex. TNFa, NFkB, and MAPK immunolabeling was increased in giant cells and dysplastic neurons. We hypothesized that an inflammatory pathway may be activated in tubers and to our surprise, numerous CD68 immunolabeled macrophages were detected in tubers, often clustered around giant cells. The expression of Fas was robust on numerous dysplastic neurons and giant cells and Fas-L was expressed by astrocytes in tubers. Caspase 8, but not caspase 2, expression was marked in numerous cell types within tubers suggesting activation of the extrinsic cell death pathway.
CONCLUSIONS: We demonstrate the potential activation of a proinflammatory cytokine pathway as well as initiation of the extrinsic cell death cascade in tubers. The presence of numerous activated macrophages in tubers and the expression of select inflammatory markers such as ICAM-1, TNFa, and MAPK suggest that activation of a proinflammatory cytokine signaling pathway may be a new and previously unappreciated feature of tubers. The selectivity of ICAM-1 expression may serve as an immunohistochemical marker to distinguish tubers from control cortex and non-TSC cortical dysplasia. These results suggest a possible relationship of tissue inflammation in tubers with epileptogenesis and may lend insights into understanding infantile spasms in TSC. Activation of the extrinisic cell death pathway suggests that a population of cell types may undergo apoptosis in tubers.
[Supported by: Tuberous Sclerosis Alliance and the Center Without Walls, MH01658, and the Esther A. and Joseph Klingenstein Fund (PBC).]