Summary Objective Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in multiple organs and severe neurological manifestations, including epilepsy, intellectual disability, and autism. Abnormalities of both neurons and astrocytes have been implicated in contributing to the neurological phenotype of TSC, but the role of microglia in TSC has not been investigated. The objectives of this study were to characterize microglial activation in a mouse model of TSC, involving conditional inactivation of the Tsc1 gene predominantly in glial cells (Tsc1GFAPCKO mice), and to test the hypothesis that microglial activation contributes to epileptogenesis in this mouse model. Methods Microglial and astrocyte activation was examined in Tsc1GFAPCKO mice by Iba1 and GFAP immunohistochemistry. Cytokine and chemokine expression was evaluated with QPCR. Seizures were monitored by video-EEG. The effect of minocycline in inhibiting microglial and astrocyte activation, cytokine expression, and seizures was tested. Results Microglial cell number and size were increased in cortex and hippocampus of 3–4 wk old Tsc1GFAPCKO mice, which correlated with the onset of seizures. Minocycline treatment prevented the increase in number and cell size of microglia in 4 week old Tsc1GFAPCKO mice. However, minocycline treatment had no effect on astrocyte proliferation and cytokine/chemokine expression and the progression of seizures in Tsc1GFAPCKO mice. Significance Microglia cell number and size are abnormal in Tsc1GFAPCKO mice, and minocycline treatment inhibits this microglia activation, but does not suppress seizures. Microglia may play a role in the neurological manifestations of TSC, but additional studies are needed in other models and human studies to determine whether microglia are critical for epileptogenesis in TSC.

Summary Objective Inhibitors of the mechanistic target of rapamycin (mTOR) pathway have antiepileptogenic effects in preventing epilepsy and pathological and molecular mechanisms of epileptogenesis in mouse models of tuberous sclerosis complex (TSC). However, long-term treatment with mTOR inhibitors may be required to maintain efficacy and potentially has chronic side effects, such as immunosuppression. Attempts to minimize drug exposure will facilitate translational efforts to develop mTOR inhibitors as antiepileptogenic agents for TSC patients. In this study, we tested intermittent dosing paradigms of mTOR inhibitors for antiepileptogenic properties in a TSC mouse model. Methods Western blot analysis of phosphorylation of S6 protein was used to assess the dose- and time-dependence of mTOR inhibition by rapamycin in control mice and conditional knock-out mice with inactivation of the Tsc1 gene in glial fibrillary acidic protein (GFAP)-expressing cells (Tsc1GFAPCKO mice). Based on the western blot studies, different dosing paradigms of rapamycin starting at postnatal day 21 were tested for their ability to prevent epilepsy or pathological abnormalities in Tsc1GFAPCKO mice: 4 days of rapamycin only (4-∞), 4 days on-24 days off (4–24), and 4 days on-10 days off (4–10). Results mTOR activity was inhibited by rapamycin in a dose-dependent fashion and recovered to baseline by about 10 days after the last rapamycin dose. 4–10 and 4–24 dosing paradigms almost completely prevented epilepsy and the 4–10 paradigm inhibited glial proliferation and megalencephaly in Tsc1GFAPCKO mice. Significance Intermittent dosing of rapamycin, with drug holidays of more than three weeks, maintains significant antiepileptogenic properties in mouse models of TSC. These findings have important translational applications in developing mTOR inhibitors as antiepileptogenic agents in TSC patients by minimizing drug exposure and potential side effects.

Microglia abnormalities may contribute to epileptogenesis in the context of neuronal involvement in TSC mouse models, but selective Tsc1 gene inactivation in microglia alone may not be sufficient to cause epilepsy, suggesting that microglia have more supportive roles in the pathogenesis of seizures in TSC.

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