Abstracts

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations of either TSC1 or TSC2 genes. In the brain, the CNS lesions most closely associated with epilepsy and autism are regions of cortical dysplasia called tubers. The Eker rat, which carries a spontaneous germline mutation of the TSC2 gene, has been studied as an animal model of TSC. Recently, we showed that early postnatal irradiation, a [ldquo]second hit[rdquo] affecting Eker TSC2 +/- cells, caused abnormal cell features which resemble some of the TSC pathologies seen in human patients. We extend our analysis in the irradiated Eker rat brain, to characterize further the neuronal and glial phenotypes of abnormal cells that might be linked to seizures.
Brain sections from irradiated and non-irradiated Eker (TSC+/-) rats were used for immunocytochemistry (ICC) of neuronal markers (NeuN, Neurofilament, MAP2, GAD67, GABA) and markers for glial and/or immature cells (GFAP, Vimentin, Nestin) using double-immunofluorescence (IF) techniques. In addition, to characterize abnormal cells that may lack functional tuberin, a phospho-ribosomal S6-protein antibody was used as an ICC marker. Individual cells located in the neocortex or in the hamartomas displaying pathological features were examined by means of light and electron microscopy (EM).
Our previous study showed that irradiation of Eker (+/-) rats generated such neuropathological features as dysmorphic (cytomegalic) neurons, giant astrocytes, and hamartoma-like lesions. Double-IF analysis combining neuronal and glial markers reveals that dysmorphic cells with neuron-like morphology also display a neuronal ICC (GFAP-negative) phenotype. At the EM level, these cells have excitatory and inhibitory synapses on soma and dendritic processes, suggesting that they are involved in neuronal circuitry. Giant astrocytes (immuno-negative for neuronal markers) are closely associated with blood vessels. Interestingly, many of these large aberrant cells demonstrate robust immunoreactivity for phospho-S6 protein expression, a marker for cells lacking tuberin. Further ICC analyses of cells in hamartomas (subcortical white matter heterotopia) reveal a mixed glio-neuronal phenotype which expresses both GFAP and GAD67/MAP2 (but not NeuN). At the EM level, phospho-S6-protein positive cells exhibit different intensities of GFAP and intermediate filament expression. These results suggest cell populations with varying degree of maturity.
Early postnatal irradiation of Eker (TSC +/-) rat pups generates some of the pathological features observed in brain lesions of TSC patients. Future studies focusing on modifying this [ldquo]second hit[rdquo] approach, and on tuberin gene pathways, will provide a better understanding how brain lesions in TSC are formed.
[Supported by: NIH NS 18895 (PAS), and NS 04502 and Tuberous Sclerosis Alliance (PBC)]