Vigabatrin inhibits seizures and mTOR pathway activation in a mouse model of tuberous sclerosis complex

Abstract

Epilepsy is a common neurological disorder and cause of significant morbidity and mortality. Although antiseizure medication is the first-line treatment for epilepsy, currently available medications are ineffective in a significant percentage of patients and have not clearly been demonstrated to have disease-specific effects for epilepsy. While seizures are usually intractable to medication in tuberous sclerosis complex (TSC), a common genetic cause of epilepsy, vigabatrin appears to have unique efficacy for epilepsy in TSC. While vigabatrin increases gamma-aminobutyric acid (GABA) levels, the precise mechanism of action of vigabatrin in TSC is not known. In this study, we investigated the effects of vigabatrin on epilepsy in a knock-out mouse model of TSC and tested the novel hypothesis that vigabatrin inhibits the mammalian target of rapamycin (mTOR) pathway, a key signaling pathway that is dysregulated in TSC. We found that vigabatrin caused a modest increase in brain GABA levels and inhibited seizures in the mouse model of TSC. Furthermore, vigabatrin partially inhibited mTOR pathway activity and glial proliferation in the knock-out mice in vivo, as well as reduced mTOR pathway activation in cultured astrocytes from both knock-out and control mice. This study identifies a potential novel mechanism of action of an antiseizure medication involving the mTOR pathway, which may account for the unique efficacy of this drug for a genetic epilepsy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. VGB treatment inhibited seizures and moderately improved survival in Tsc1 GFAPCKO mice.

(A) Representative EEG recordings of Tsc1GFAPCKO mice treated with vehicle or vigabatrin. (B) Seizures started to develop in vehicle-treated Tsc1GFAPCKO mice (Fig. 1A, KO + Veh) around 3 weeks and became progressively more frequent with age. VGB treatment (KO + VGB) almost completely suppressed the development of seizures in Tsc1GFAPCKO mice (*p<0.05 by one-way ANOVA, n = 13 mice/group). (C) Survival analysis showed that vehicle-treated Tsc1GFAPCKO mice die prematurely with 50% mortality around 7 weeks of age and 100% mortality by 11 weeks. VGB treatment modestly improved the survival of Tsc1GFAPCKO mice compared to the vehicle treated Tsc1GFAPCKO mice (*p<0.05 by Chi-Square test, comparing the two groups, n = 13 mice/group), but all VGB-treated mice still died by age of 14 weeks. KO = Tsc1GFAPCKO mice, Veh  =  vehicle, VGB  =  vigabatrin.

Figure 2. VGB treatment increased brain GABA concentrations in Tsc1 GFAPCKO and control mice.

GABA levels were measured using a commercial mouse GABA ELISA kit. VGB treatment for one week increased the GABA levels in both neocortex (A) and hippocampus (B) of Tsc1GFAPCKO mice (KO+VGB), compared with the vehicle-treated Tsc1GFAPCKO group (KO+Veh). Similar effects of VGB were observed in control mice in hippocampus only (Cont+VGB versus Cont+Veh). Data were derived from three separate experiments. *p<0.05, **p<0.01 versus vehicle-treated mice by two-way ANOVA (n = 6–7 mice/group). Cont  =  control mice, KO  =  Tsc1GFAPCKO mice, Veh  =  vehicle, VGB  =  vigabatrin.

Figure 3. VGB treatment decreased the number of GFAP positive cells in hippocampus of Tsc1 GFAPCKO mice.

(A) Vehicle-treated Tsc1GFAPCKO mice (KO + Veh) displayed a diffuse increase in GFAP-positive cells in neocortex and hippocampus compared with the vehicle-treated control mice (Cont + Veh). VGB treatment partially prevented this increase in GFAP-positive cells in Tsc1GFAPCKO mice (KO + VGB). (B) Quantitative analysis demonstrated a 2–2.5 fold increase in GFAP-positive cell in vehicle-treated Tsc1GFAPCKO group (KO + Veh) compared with vehicle-treated control group (Cont + Veh) in neocortex (CT), dentate gyrus (DG) and CA1 of hippocampus. #p<0.05, ## p<0.01, ### p<0.001 versus vehicle-treated control mice by two-way ANOVA (n = 4–5 mice/group). VGB treatment decreased GFAP-positive cells in Tsc1GFAPCKO mice (KO+VGB). **p<0.01 versus vehicle-treated Tsc1GFAPCKO mice by two-way ANOVA (n = 4–5 mice/group). Scale bars = 500 µm. Cont  =  control mice, KO  =  Tsc1GFAPCKO mice, Veh  =  vehicle, VGB  =  vigabatrin, CT  =  neocortex, DG  =  dentate gyrus, CA1  =  CA1 pyramidal cell layer of hippocampus.

Figure 4. VGB treatment did not prevent neuronal disorganization in Tsc1 GFAPCKO mice.

The left panels show Cresyl violet staining of a brain section at low (upper) and high (lower) magnification of vehicle-treated control mice (Cont + Veh). Vehicle-treated Tsc1GFAPCKO group (KO + Veh) exhibited widely dispersed pyramidal cell layers (arrows in the middle panels) in all regions of hippocampus (CA1–CA4) compared with control mice. As shown in the right panels, VGB treated Tsc1GFAPCKO mice (KO + VGB) had a similar pattern as vehicle-treated Tsc1GFAPCKO group, with no apparent effect on this neuronal disorganization (arrows in right panel). Scale bar = 500 µm. Cont  =  control mice, KO  =  Tsc1GFAPCKO mice, Veh  =  vehicle, VGB  =  vigabatrin.

Figure 5. VGB decreased activation of the mTOR pathway in vivo.

(A, B) Western blotting shows P-S6 (Ser240/244), total S6, and beta-actin expression in neocortex (A) and hippocampus (B) of control mice and Tsc1GFAPCKO mice treated with vehicle or VGB at daily doses of 50, 100 and 200 mg/kg for 1 week. Quantitative summary demonstrates that vehicle-treated Tsc1GFAPCKO mice have significantly increased P-S6 levels compared with control mice (# p<0.05, ### p<0.001 versus control mice by two-way ANOVA, n = 5–9 mice/group), and VGB inhibited the activation of S6 in Tsc1GFAPCKO mice in a dose-dependent fashion (* p<0.05 versus vehicle-treated Tsc1GFAPCKO mice by two-way ANOVA, n = 5–9/group). The ratio of P-S6/total S6 was normalized to the vehicle-treated Tsc1GFAPCKO group. (C, D) Western blotting shows P-S6 (Ser240/244), total S6 and beta-actin expression in non-KO control mice administered vehicle or VGB at 50, 100 and 200 mg/kg/day for 1 week starting at age of three weeks. Quantitative summary demonstrates that VGB inhibited the activation of S6 in control mice in a dose-dependent fashion. The ratio of P-S6/total S6 was normalized to the vehicle-treated control group. *p<0.05, **p<0.01, *** p<0.001 versus vehicle-treated non-KO control mice by one-way ANOVA (n = 5–9 mice/group). Cont  =  control mice, KO  =  Tsc1GFAPCKO mice, Veh  =  vehicle.

Figure 6. VGB decreased activation of the mTOR pathway in vitro.

A) Western blotting shows P-S6 (Ser 240/244), total S6, and beta-actin expression in primary cultured astrocytes derived from Tsc1GFAPCKO and non-KO control mice. Vehicle or VGB at a dose of 0.06, 0.3 and 0.6 mM was added to the culture medium for 16 hours. Overall, Tsc1GFAPCKO astrocytes showed increased P-S6 expression compared with astrocytes from control mice. VGB blocked the activation of P-S6 in a dose-dependent fashion in both control and KO astrocytes. The ratio of P-S6/total S6 was normalized to the vehicle-treated control group (Cont) or vehicle-treated Tsc1GFAPCKO group (KO). Quantitative summary demonstrates that VGB treatment at doses of 0.3 and 0.6 mM significantly inhibits the activation of P-S6 in astrocytes of both Tsc1GFAPCKO and control mice. B) In contrast to VGB, phenobarbital had no effect on P-S6 expression in control astrocytes at doses that are effective in potentiating GABA-mediated inhibition . *p<0.05, **p<0.01 versus Veh by one-way ANOVA (n = 8 mice/group). Cont  =  control mice, KO  =  Tsc1GFAPCKO mice, Veh  =  vehicle.