Abstracts

Rationale: Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in the TSC1 or TSC2 tumor suppressor genes. Epilepsy, cognitive impairments, and autism are the most common neurological manifestations of this disease. Numerous studies have implicated pro-inflammatory mechanisms as contributing factors to neurological dysfunction in TSC, as gene and protein expression studies have demonstrated robust immune activation in human TSC cortical lesions. One limitation of these studies is that they cannot distinguish between cause and effect, nor reveal any time-dependent processes. We previously demonstrated in a mouse model of TSC that there is elevated GFAP expression during the period when chronic epilepsy is well established. This is also associated with enhanced IL-1ߠsignaling (Coley et al., AES 2014: Abst. 3.041). Here we sought to evaluate whether any of these changes may already be occurring during the latent period or the pre-epileptic stage. Methods: Control and Tsc1 mice (Tsc1cc Nestin-rtTA+ tet-OP-cre+ E13doxy) were sacrificed at P7-8 and P15-16, a time window during which no seizures occur in this model. Frozen and fixed whole brains were used for Western blotting and immunohistochemistry (n=4-8/genotype/time point). Blots were probed with the mammalian target of rapamycin complex 1 (mTORC1) activation marker P-S6 (ser235/236), the astrocytic marker GFAP, the interleukins IL-1a and IL-1߬ and the IL1 receptor antagonist (IL1Ra). Beta-actin was used to control for differences in protein loading. P-S6 (ser235/236) values were expressed as ratios to total S6. Statistical significance (p < 0.05) was assessed using two-tailed t-tests. Paraformaldehyde-fixed brains were sectioned and immunolabeled with P-S6 (ser235/236), GFAP, and the immediate early gene c-Fos. Results: We found increased P-S6 (ser235/236), IL-1a and IL1Ra levels in the Tsc1 mice already at P7-8, however the differences between genotypes became significant at P15-16 (324.3% of control, p < 0.0001 for P-S6 (ser235/236p); 166.5%, p < 0.01 for IL-1a; 157.8%, p < 0.01 for IL1Ra). GFAP levels were comparable to controls at P7-8, but were significantly upregulated at P15-16 (203.2% of control, p < 0.05). In contrast, there were no significant changes in IL-1ߠexpression at both time points studied. Immunohistochemical analysis confirmed prominent activation of mTORC1 in astrocytes at both time points. Focal neocortical astrocytosis was already observed at P7-8 in the Tsc1 mice. At P15-16, the astrocytosis became more diffuse. We also observed dramatic changes in astrocytic morphology, such as enlarged cell bodies and shorter processes, which is consistent with loss of normal astrocytic function. We did not detect any concurrent c-Fos staining at either age, suggesting that astroglial activation could not be attributed to altered neuronal function. Conclusions: This study provides evidence for early astrocyte-mediated inflammatory responses in TSC, and suggests that Tsc1-deficient astrocytes may become more dysfunctional over time. `Our data also suggest the possibility of preventing epilepsy in TSC by appropriate modulation of these early responses. Funding: Supported by: University Research Foundation