Abstracts

Rationale: Tuberous sclerosis complex (TSC) is a genetic disorder, caused by mutations in the TSC1 or TSC2 genes, which leads to hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) pathway and stimulates cell growth and proliferation, promoting tumor growth. Epilepsy occurs in up to 90% of TSC patients and is one of the most disabling symptoms of this disease, but the mechanisms of epileptogenesis in TSC are poorly understood. Microglia, the resident macrophages of the brain, act as the primary form of active immune defense and inflammatory responses in the CNS, and also regulate other aspects of basic brain development and function, such as synaptic transmission and plasticity. Microglia are activated in response to a variety of brain insults, including ischemia, trauma, infection, and epilepsy, as well as in tuber specimens from TSC patients. However, the pathophysiological role of microglia in the neurological manifestations of TSC is relatively unexplored. In this study, we tested the role of microglia in epileptogenesis in a novel mouse model of TSC, Tsc1-CX3CR1 CKO mice. Methods: Four week old Tsc1-CX3CR1 CKO mice, which involve conditional inactivation of the Tsc1 gene primarily in microglia, and littermate control mice were used in these experiments. Immunohistochemistry to the microglia marker Iba1 was used to evaluate microglial activation. Western Blot analysis was used to evaluate protein expression of the ratio of P-S6/S6, an indicator of mTOR1 activation. Video-EEG was used for monitoring seizures. Some mice were treated with rapamycin, an inhibitor of mTOR1 activation, for 1 wk for Western blot and up to 8 wks for video-EEG monitoring of seizures. Results: Iba1 immunohistochemistry showed that Iba1 expression was increased in cortex and hippocampus of 4 wk old Tsc1 CX3CR1 CKO mice compared with control mice. Western blotting showed that the ratio of P-S6/S6 was significantly increased in Tsc1-CX3CR1 CKO mice compared with control mice. Rapamycin treatment at the dose of 3 mg/kg/day for 1 wk reversed the increased P-S6/S6 ratio (n=8 mice per group, p < 0.01). Video-EEG monitoring demonstrated seizures in the majority of vehicle-treated Tsc1-CX3CR1 CKO mice (n=5 of 9 mice), which were inhibited in rapamycin-treated mice. Conclusions: Microglial activation and seizures developed in Tsc1-CX3CR1 CKO mice. Rapamycin treatment inhibited mTOR1 activation and seizure development, indicating that microglia play an important role in epileptogenesis in Tsc1-CX3CR1 CKO mice. Funding: NIH R01 NS056872