EMBRYONIC NEURONAL MARKERS IN TUBEROUS SCLEROSIS - SINGLE-CELL MOLECULAR PATHOLOGY

One hallmark of tuberous sclerosis (TSC) is the presence of highly epi leptogenic dysplastic cerebral cortex (tubers) composed of abnormally shaped neurons and giant cells, Mutation of the TSC gene (TSC2) may di srupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferat ion, To test the hypothesis that cells in tubers mag. retain the molec ular phenotype of embryonic or immature neurons, tubers from five TSC patients were probed with antibodies to proteins expressed in neuronal precursors (nestin, Ki-67, and proliferating cell nuclear antigen), M any dysmorphic neurons and giant cells in tubers were stained by these antibodies, while neurons in adjacent normal and control cortex were not labeled, To further characterize the molecular phenotype of cells in tubers, we developed a methodology in which poly(A)(+) mRNA was amp lified from immunohistochemically labeled single cells in paraffin-emb edded brain specimens, This approach enabled us to detect mRNAs encodi ng nestin, and other cytoskeletal elements, cell cycle markers, and sy nthetic enzymes present in individual nestin-stained cells by means of reverse Northern blotting, We conclude that the presence of immature phenotypic markers (mRNAs and proteins) within tubers suggests disrupt ion of cell cycle regulation and neuronal maturation in TSC during cor tical development, Characterization of multiple mRNAs within fixed, im munohistochemically labeled cells provides a powerful tool far studyin g gene expression and the molecular pathophysiology of many neurologic diseases.