Abstracts

Rationale: Tuberous Sclerosis Complex (TSC) is a multisystem autosomal dominant disease caused by inactivating mutations in TSC1 or TSC2 genes. The most common neurological symptoms in TSC include treatment-resistant epilepsy, mental retardation and autism. There is now accumulating evidence that pro-inflammatory cytokines, produced and released mainly by reactive astrocytes and activated microglia, are implicated in a wide range of chronic neurological disorders, including refractory epilepsy. The purpose of this study was to evaluate the degree of inflammation in TSC brain lesions in human, with the hypothesis that activated glial cells and pro-inflammatory cytokines will be increased in both cortical tubers and peri-tuberal brain (PTB), possibly contributing to widespread network dysfunction. Methods: Human TSC specimens (n=10; ages 0-7 years) were prospectively collected following epilepsy surgery at NYULMC. Region-matched control autopsy brain samples from cases with normal neurological history were obtained from the Maryland Brain and Tissue Bank (n=7; ages 0-8 years). Tissue samples were collected and handled in accordance with the NYULMC Institutional Review Board. Fresh frozen specimens were used to prepare protein extracts for Western blotting. Blots were probed with the astrocytic and microglial markers GFAP and Iba1, as well as with the pro-inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). To determine statistical significance (p<0.05), one-way ANOVA followed by Student's t-test were used. Results: GFAP and Iba1 levels were significantly elevated in both tubers (343±115% of control, n=5; p<0.05 for GFAP, and 346±35% of control, n=5; p<0.0001 for Iba1) and PTB (225±37% of control, n=5; p<0.001 for GFAP, and 247±37% of control, n=5; p<0.001 for Iba1). The inactive pro-IL-1β was highly upregulated in tubers (319±114% of control, n=6; p<0.05) and in PTB (228±70% of control, n=4; p<0.001), and this was accompanied by a commensurate increase in the secreted, active form of IL-1β (192±28% of control in tubers, n=6; p<0.05, and 159±17% of control in PTB, n=4; p<0.05). Pro-TNF-α expression was similarly upregulated in both tubers and PTB (289±34% of control, n=6, and 204±4% of control n=4, respectively; p<0.05), but the levels of secreted TNF-α were only moderately increased (149±24% of control in tubers, n=6, and 117±4% of control in PTB, n=4; p>0.05). Conclusions: Our data demonstrate robust increases in pro-inflammatory markers in TSC lesions, which extend beyond the cortical tuber borders, suggesting a potential pathogenic role of neuroinflammation in epileptogenesis, cognitive dysfunction and altered social behavior in TSC patients. Therapies that suppress glial activation, or directly block cytokine signaling, might be useful to ameliorate the complex neurological deficits in these patients. Acknowledgments: Shaw Foundation, FACES, Tuberous Sclerosis Alliance, NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD, contract HHSN275200900011C.