The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy

Abstract

Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Figure 1.

Kainate-induced seizures results in biphasic activation of the mTOR pathway. A, In the acute phase of kainate status epilepticus, Western blotting shows P-S6 and total S6 expression in hippocampus (top; Hip) and neocortex (bottom) at different time intervals within the first 24 h after the onset of seizures (which lasted 6.0 ± 1.3 h). B, Quantitative summary demonstrates that phosphorylation of S6 was increased within 1 h of the onset of kainate-induced seizures, peaked at 3 h, and returned to baseline after 6 h in both hippocampus and neocortex. The ratio of P-S6/S6 was normalized to rats without kainate (0 h). C, In the chronic phase after kainate status epilepticus, Western blotting shows P-S6 and total S6 expression in hippocampus (top) and neocortex (bottom) at longer time intervals over several weeks. D, Quantitative summary demonstrates that phosphorylation of S6 increased again at 3 d in hippocampus, but not neocortex, peaked at 5 d, and decreased to baseline by 5 weeks. The ratio of P-S6/S6 was normalized to rats without kainate (0d). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA (n = 6 rats for each time point and group).

Figure 2.

Rapamycin pretreatment abolishes mTOR activation from kainate-induced seizures both acutely and chronically but does not alter the acute properties of kainate status epilepticus. A, In the acute phase of kainate status epilepticus, Western blotting shows P-S6 and S6 expression at different time interval after kainate-induced status epilepticus in rapamycin-pretreated (KA+, Rap+) and vehicle-pretreated (KA+, Rap−) rats in hippocampus and neocortex. Vehicle-treated rats that did not receive kainate (KA−, Rap−; 0 h) serve as an additional control. B, In the chronic phase after kainate status epilepticus, Western blotting shows P-S6 and total S6 expression at longer time intervals over several weeks in rapamycin- and vehicle-pretreated rats in hippocampus. Note that pretreatment with rapamycin almost completely inhibits the activation of P-S6 both acutely and chronically after kainate status epilepticus (n = 6 rats for each time point and group). C, Rapamycin pretreatment had no effect on the latency, severity (stage), or duration of kainate-induced status epilepticus based on behavioral analysis (n = 10 rats per group), as well as ictal EEG spike discharges (see Results). Veh, Vehicle.

Figure 3.

Rapamycin pretreatment decreases neuronal cell death after kainate-induced seizures. Representative sections of Fluoro-Jade B staining in hippocampus of vehicle- (Veh+KA; A–C) and rapamycin-pretreated (Rap+KA; D–F) rats are shown 7 d after kainate status epilepticus. Abundant Fluoro-Jade B-positive neurons can be found in vehicle-pretreated rats in CA1, CA3, and hilus but not in rapamycin-pretreated rats. G, Quantitative analysis demonstrates a significant decrease in Fluoro-Jade B-positive neurons in rapamycin-pretreated rats. Scale bar, 200 μm. *p < 0.05, ***p < 0.001 by t test (n = 10 rats per group).

Figure 4.

Rapamycin pretreatment decreases neurogenesis after kainate-induced seizures. Representative sections show BrdU staining in control (Cont) rats that did not receive kainate (A; KA) and vehicle-pretreated (B; Veh) and rapamycin-pretreated (C; Rap) rats that received kainate. D, Quantitative analysis demonstrates a significant decrease in BrdU-positive cells in the dentate gyrus of rapamycin-pretreated rats compared with vehicle-pretreated rats after kainate status epilepticus. Scale bar, 200 μm. ***p < 0.001 by one-way ANOVA (n = 3 in control rats; n = 6 in kainate-treated rats).

Figure 5.

Rapamycin pretreatment reduces mossy fiber sprouting after kainate-induced seizures. Timm's staining shows mossy fiber staining from control (Cont) rats that did not receive kainate (A; KA) and vehicle-pretreated (B; Veh) and rapamycin-pretreated (C; Rap) rats 28 d after kainate-induced status epilepticus. A1–C1 are higher magnification of boxed regions in A–C, respectively. m, Molecular layer; g, granule cell layer. D, Quantitative analysis demonstrates a significant decrease in Timm's score in rapamycin-pretreated rats compared with vehicle-pretreated rats after kainate-induced status epilepticus. Scale bars: A–C, 200 μm; A1–C1, 50 μm. ***p < 0.001 by one-way ANOVA (n = 3 in control rats; n = 6 in kainate-treated rats).

Figure 6.

Rapamycin pretreatment reduces KA-induced epilepsy in rats. A, Experimental design demonstrating timing of drug treatment and video-EEG monitoring. B, Representative EEG background and interictal epileptiform spikes in vehicle- and rapamycin-pretreated rats. Note there are significantly more epileptiform spikes in vehicle-pretreated rats. **p < 0.01, ***p < 0.001 by t test. C, Number of spontaneous seizures per day in vehicle- and rapamycin-pretreated rats during the first week after status epilepticus. D, Representative electrographic seizure is shown (top). Rapamycin postponed spontaneous seizure onset and reduced seizure frequency. Seizures started to develop within 1–2 weeks after kainate administration and became more frequent in vehicle-pretreated rats, whereas only rare seizures occurred in rapamycin-pretreated rats. *p < 0.05 by two-way ANOVA (n = 6–8 rats per time point and group). KA, Kainate; Rap, rapamycin; Veh, vehicle.

Figure 7.

Rapamycin posttreatment after status epilepticus blocks the late phase on mTOR activation and reduces mossy fiber sprouting and epilepsy but not cell death. A, B, Rats were treated with vehicle (KA+, Rap−) or rapamycin (KA+, Rap+) starting 24 h after onset of kainate status epilepticus for 6 consecutive days. As assayed at 1 week after status epilepticus, rapamycin posttreatment blocked the late phase of mTOR activation. In contrast, as an additional control, phenobarbital, which can suppress acute seizure activity, had no effect of kainate-induced mTOR activation (KA+, PB+). *p < 0.05 compared with KA+Veh and KA+PB by one-way ANOVA (n = 4 rats per group). C–E, Rapamycin posttreatment (KA+Rap) reduced mossy fiber sprouting and Timm's score in dentate gyrus after kainate status epilepticus compared with controls (KA+Veh). *p < 0.05 by t test (n = 6 rats per group). F, Rapamycin posttreatment had no significant effect on neuronal death in hippocampus after kainate status epilepticus. G, Rapamycin posttreatment reduced frequency of spontaneous seizures up to 6 weeks after kainate status epilepticus. *p < 0.05 by one-way ANOVA (n = 7 rats per time point for rapamycin group; n = 7–10 rats per time point for vehicle group).