Abstracts

Balloon cells (BCs) in focal cortical dysplasia (FCD) and giant cells (GCs) in tubers of the tuberous sclerosis complex (TSC) share phenotypic similarities. TSC1 or TSC2 gene mutations in TSC lead to mTOR pathway activation and result in phosphorylation of p70S6kinase (phospho-S6K) and ribosomal S6 (phospho-S6) proteins. We hypothesized that downstream components of the mTOR pathway are activated in FCD and that BCs and GCs exhibit distinct gene expression profiles. FCD tissue specimens (n=10; mean age=12.3 years, 5 females, 5 males) and tubers (n=10, mean age=8.25 years, 4 males and 6 females) resected for the treatment of medically intractable epilepsy were analyzed by immunohistochemistry and single cell gene expression analysis. Sections were probed with either NeuN (1:100, Chemicon), phospho-S6 (Ser235/236; 1:200 dilution), phospho-S6K (Thr389; 1:200 dilution), 4EBP1 (Tyr 305, 1:100; all from Cell Signaling, New England Biolabs, Beverly, MA) or STAT3 (1:250, LabVison, Fremont, CA) antibodies. Amplified, radiolabeled mRNA from single phospho-S6 labeled BCs, GCs, or NeuN labeled control neurons was used to probe cDNA arrays containing candidate genes including cell signaling molecules, cell adhesion molecules, growth factors/receptors, and transcription factors. TSC genotyping on genomic DNA extracted from the tubers revealed 7 TSC2 and 3 TSC1 mutations. Selective expression of phospho-S6K and phospho-S6 was detected immunohistochemically in GCs whereas only phospho-S6 was observed in BCs. Two proteins activated by phospho-S6K, phospho-STAT3, and phospho-4EBP1, were detected in GCs but not BCs. Among 60 candidate genes assayed in phospho-S6 immunolabeled BCs and GCs, differential expression of 24 mRNAs distinguished BCs, GCs, and control neurons. Only 4 genes showed similar expression profiles between BCs and GCs. The observed changes in expression of BF-1, BMP-6, EGFR, CaMKII, CREB, IGF-1, IGF-2, OTX-1, TGFRbeta1, and TGFRbeta3 mRNAs represent the first report of altered transcription of these genes in FCD. Tuberin mRNA levels were reduced in GCs from TSC patients with identified TSC2 gene mutations but were unchanged in BCs. Phospho-S6K, -S6, -STAT3, and -4EBP1 expression in GCs results from loss of hamartin-tuberin mediated mTOR pathway inhibition whereas phospho-S6 expression in BCs does not support mTOR cascade activation in FCD. Differential gene expression profiles found in BCs and GCs supports the hypothesis that these cell types derive by distinct pathogenic mechanisms. (Supported by NINDS R010405, R21-NS39938, R21NS40231, Parents Against Childhood Epilepsy (PACE). )