Abstracts

Rationale: Tuberous Sclerosis Complex (TSC), a multisystem autosomal dominant disorder due to mutations in TSC1 and TSC2 genes, often manifests with abnormal brain development, therapy-resistent epilepsy, mental retardation and autism. We have demonstrated robust immune activation in human TSC cortical lesions (Dilsiz et al., AES 2012: Abst. 3.046), including elevated expression of pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), both previously implicated in epileptogenesis. Here, our objective was to evaluate the activation status of pro-inflammatory cytokine in a mouse model of TSC in order to identify new therapeutic targets for epilepsy prevention. Methods: Tsc1cc Nestin-rtTA+ tet-OP-cre+ mice were produced by timed inactivation of Tsc1 in neuronal progenitor cells at E12.5-13.5. (PNAS 2011; 108: E1070-9). Mutant and control mice were sacrificed at P30-32 (n=6/genotype) and frozen whole brains were used to prepare whole cell and membrane protein fractions. Western blots were probed with astrocytic and microglial markers GFAP and Iba-1; IL-1β; IL1 receptor antagonist (IL1Ra); IL1 receptor I (IL1R1); and the nuclear factor kappa-B (NF-κB) subunits p50 and p65. Beta actin was used to control for differences in protein loading. The effect of IL-1β on neuronal excitability was assesed by measuring spontaneous excitatory postsynaptic currents (sEPSCs) in cortical slices. Recombinant IL-1β (10ng/ml) was bath applied for 30 min. Recordings were performed in layer V pyramidal neurons in ex vivo cortical slices from P45 control and Tsc1 mice (n=3-4/genotype), at a holding potential of -60mV. Statistical significance (p<0.05) was assessed using t-tests. Results: We found significant increases in GFAP (p<0.05), Iba1 (p<0.05), IL-1β (p<0.0001), IL1R1 (p<0.01), NF-κB p50 (p<0.05) and p65 (p<0.05), accompanied by a significant reduction in IL-1Ra (p<0.05) levels in Tsc1 mutant mice relative to controls. Bath application of recombinant IL-1β induced a significant increase in AMPA receptor-mediated sEPSC amplitude (p<0.05) in slices from Tsc1 mice, but no noticeable changes in layer V pyramidal neurons from wild-type controls (p>0.05). Conclusions: Our results demonstrate elevated IL-1β responses in the Tsc1 mouse brain and suggest a potential role for altered NF-κB function in mediating these responses. Our data also support the validity of this animal model to further evaluate the functional interactions between pro-inflammatory pathways, neuronal dysfunction and epileptogenesis, and justify future studies to investigate the therapeutic potential of immune modulators to improve neurological deficits in TSC. Supported by: NIH/NINDS R21 NS080268