Abstracts

Rationale: Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease due to loss of TSC1 or TSC2 function and resulting in altered mTOR signaling with subsequent abnormalities in brain development. TSC manifests with cognitive dysfunction, autism and early onset epilepsy, which is frequently refractory and requires surgical therapy. While earlier studies have focused mainly on characterization of cortical tubers, in this study, we sought to analyze in greater detail the unique molecular and cellular features of the ictogenic peri-tuberal brain (PTB), as compared with cortical tuber and control specimens. Methods: Human TSC specimens (n=20; ages 0-10 years) were collected prospectively during resective epilepsy surgery at NYUMC. Age- and region-matched post mortem samples from cases with no neurological symptoms obtained from the Maryland Brain and Tissue Bank were used as controls (n=5). This study was approved by the Institutional Review Board at NYUMC. Western blotting was used to assess the expression of the mTORC1 and mTORC2 biomarkers pS6 (Ser235/236), pS6 (Ser240/244) and pAkt (Ser473). In addition, the axonal markers SMI 312 and growth associated protein (GAP)-43, as well as myelin basic protein (MBP) were quantified to characterize axonal growth and white matter development. Paraformaldehyde-fixed specimens were sectioned and immunolabeled with neuronal filament markers (SMI311, SMI312), glial markers (vimentin, MBP) and pS6 (Ser235/236). One-way ANOVA was used to establish statistical significance (p<0.05). Results: mTORC1 activity was increased in tubers (n=4) and PTB (n=3), as demonstrated by increased pS6/S6 ratios normalized to control: 275% in tubers and 191% in PTB for pS6 (Ser235/236), p<0.001; 168% in tubers and 144% in PTB for pS6 (Ser240/244), p<0.05 . Similarly, tubers (n=4) and PTB (n=3) showed decreased mTORC2 activity, as demonstrated by lowered pAkt/Akt ratios relative to control (57% in tubers and 67% in PTB, p<0.0001). Consistent with the Western blot results, we found widespread pS6-positive giant cells and dysplastic neurons in tubers and PTB. Expression of the axonal marker SMI312 and of GAP-43, a marker for active growth cones, were both increased in tubers (n=6) and PTB (n=7) relative to control (205% in tubers and 128% in PTB for SMI312, p<0.001; and 178% in tubers and 146% in PTB for GAP-43, p<0.001). This pattern correlated with reduced MBP expression relative to control (29% in tubers, n=6 and 40% in PTB, n=7, p<0.005). Conclusions: Our results demonstrate an extension of abnormal cell types in the PTB and demonstrates, for the first time, aberrant axonal development in human TSC lesions. This data supports the hypothesis that both cellular dysplasia and altered connectivity may underlie the broad spectrum of neurological deficits in TSC patients, including seizure generation and epileptogenesis. Acknowledgments: FACES, Tuberous Sclerosis Alliance, NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD, contract HHSN275200900011C.