Expression of the anti-apoptotic protein survivin amd altered function of IGF-1 pathway in TSC2 brain tubers

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome characterized by multiorgan development of benign and occasional malignant tumors. The neurological manifestations include epilepsy, developmental delay, mental retardation, and autism. The tubers of TSC are focal lesions occurring predominantly at the gray-white matter boundary, characterized by the loss of normal cytoarchitecture, with presence of abnormally enlarged and dysmorphic neurons, giant and glial cells. These abnormalities reflects dysregulation of cell growth, differentiation, and migration processes. Some morphological features of TSC cortical tubers are comparable to those observed in focal cortical dysplasia (FCD), a frequent finding in patients with chronic intractable epilepsy. The focal cortical dysplasia of Taylor’s balloon cell type (FCDbc) is a subtype of FCD. Our study comprises cortical tubers obtained from 9 patients who met clinical criteria for tuberous sclerosis, epileptogenic areas were also obtained from 2 patients with chronic intractable epilepsy displaying FCDbc and 2 with frontal cortex epilepsy without a history of TSC. Control cortex were from 7 patients not affected by TSC or other forms of epilepsy. All the exons of TSC1 and TSC2 genes were analyzed in the patient with FCDbc, and no significant sequence alterations were found. The dysmorphic neurons of the tuber upper layer displayed immunoreactivity for neurofilaments, while the round smaller cells of the deeper layers were devoid. All neural cells throughout the tuber were positive for nestin. In FCDbc and frontal cortex all neural cells were positive for neurofilaments and none for nestin. The other neural marker III  tubulin was also absent in tubers. While there was a generalized positivity to phosphorylated S6K1 and S6 in tubers and FCDbc, in frontal cortex only few cells did. We have previously reported that human isolated and heterozygous TSC2+/- epithelioid cells (positive for keratine 8/18 and HMB45) express survivin, an inhibitor of apoptosis protein family, while TSC2-/- smooth muscle cells do after exposure to IGF-1. The majority of brain lesions of TSC patients is heterozygous. TSC2 tubers strongly express survivin while in FCDbc only few cells were positive. Non-TSC frontal cortex did not show any staining for survivin. Phosphorylation of Akt and S6 were induced by IGF-1 exposure and inhibited by the specific PI3K inhibitor LY294002 (LY) in control frontal cortex and FCDbc, but not in TSC-cortical tubers. Erk expression was comparable in control frontal cortex and tubers, and was not affected by any treatment. Phosphorylated form of Erk was undetectable even after IGF-1 exposure in control cortex, while Erk was strongly phosphorylated in TSC tubers and FCDbc; IGF-1 further increased the extent of such activation. LY antagonized IGF-1-mediated Erk phosphorylation in TSC tubers and FCDbc. In agreement with previous report we show that neural cells of TSC tubers are biologically made up by at least two type of cells, those situated in the upper layers morphologically abnormal but showing the typical neuronal markers and those more deeper round and marked by nestin. All of them show elevated levels of survivin as the TSC2+/- epytheliod cells do. We propose that the expression of survivin may contribute to perturbation of cortical differentiation in tubers probably through the inhibition of the developmental neural cell death and facilitation of cell division. In addition, the growth factor pathways seem altered so that the activation of IGF-1 receptor leads to the phosphorylation of Erk rather than Akt. This also may contribute to the abnormal proliferation and cortical differentiation occurring in TSC tubers.