Impaired autophagy in neurons after disinhibition of mammalian target of rapamycin and its contribution to epileptogenesis

Abstract

Certain mutations within the mammalian target of rapamycin (mTOR) pathway, most notably those affecting the tuberous sclerosis complex (TSC), lead to aberrant activation of mTOR and result in a high incidence of epilepsy in humans and animal models. Although hyperactivation of mTOR has been strongly linked to the development of epilepsy and, conversely, inhibition of mTOR by rapamycin treatment is protective against seizures in several models, the downstream epileptic mechanisms have remained elusive. Autophagy, a catabolic process that plays a vital role in cellular homeostasis by mediating the turnover of cytoplasmic constituents, is negatively regulated by mTOR. Here we demonstrate that autophagy is suppressed in brain tissues of forebrain-specific conditional TSC1 and phosphatase and tensin homlog knock-out mice, both of which display aberrant mTOR activation and seizures. In addition, we also discovered that autophagy is suppressed in the brains of human TSC patients. Moreover, conditional deletion of Atg7, an essential regulator of autophagy, in mouse forebrain neurons is sufficient to promote development of spontaneous seizures. Thus, our study suggests that impaired autophagy contributes to epileptogenesis, which may be of interest as a potential therapeutic target for epilepsy treatment and/or prevention.

Figure 1.

TSC1KO and PTENKO mice demonstrate spontaneous recurrent seizures and increased mortality. a, Percentage of WT (circle), TSC1KO (square), and PTENKO (triangle) mice that displayed behavioral seizure activities by 10 weeks of age. b, Percentage of survival of wild-type, TSC1KO, and PTENKO mice by 10 weeks of age. c, d, Representative electrographic recordings in TSC1KO and PTENKO mice during a spontaneous seizure episode. e, Western blot showing increased p-S6 levels in TSC1KO mouse brains at postnatal week 4. f, Quantification of p-S6. *Significant difference between WT and TSC1KO mice on day 28 (mean ± SEM; n = 3; p < 0.05, ANOVA). ^#Significant difference between day 28 and day 14 or 21 in WT mice (mean ± SEM; n = 3; p < 0.05, ANOVA). A.U., Arbitrary units.

Figure 2.

Autophagy levels are reduced in TSC1KO mice. a, Representative Western blots from lysates prepared from cortex, hippocampus, and cerebellum of wild-type mice, TSC1KO mice, and TSC1KO mice treated with rapamycin (TSC1KO/R) (5 mg/kg, i.p.) for 3 d. b, Quantifications of phospho-Ulk1 against total Ulk1. c, Quantifications of phospho-S6 against total S6 (ANOVA with Tukey's post hoc test; n = 5; *p < 0.05; **p < 0.01; ***p < 0.001, between TSC1KO and wild-type or TSC1KO/R). d, Representative Western blot of hippocampal lysates of wild-type and TSC1KO mice prepared from slices incubated in ACSF or CQ (100 μm) for 2 h. e, Quantification of autophagy flux, as measured by the LC3II/LC3I ratio in the CQ-treated slices over that in ACSF-treated slices, revealed a significant reduction in TSC1KO mice (mean ± SEM; n = 5; t test, p < 0.05). Ctx, Cortex; Hip, hippocampus; Crb, cerebellum; Veh, vehicle.

Figure 3.

Autophagy levels are reduced in PTENKO mice. a, Representative Western blots from lysates prepared from cortex, hippocampus, and cerebellum of wild-type mice, PTENKO mice, and PTENKO mice treated with rapamycin (PTENKO/R) (5 mg/kg, i.p.) for 3 d. b, Quantifications of phospho-Ulk1 against total Ulk1. c, Quantifications of phospho-S6 against total S6 (ANOVA with Tukey's post hoc test, *p < 0.05; **p < 0.01; ***p < 0.001, between PTENKO and wild-type or PTENKO/R; ^#Significant difference between wild-type and PTENKO/R). d, Representative Western blot of hippocampal lysates of wild-type and PTENKO mice prepared from slices incubated in ACSF or CQ (100 μm) for 2 h. e, Quantification of autophagy flux, as measured by the LC3II/LC3I ratio in the CQ-treated slices over that in ACSF-treated slices, revealed a significant reduction in PETNKO mice (mean ± SEM; n = 5; t test, p < 0.05). Ctx, Cortex; Hip, hippocampus; Crb, cerebellum; Veh, vehicle.

Figure 4.

Autophagy is inhibited in the brains of TSC patients. a, Representative Western blots from control cortex from normal human brains compared with cortical tuber or SEGA tissues from human TSC patients. Quantifications of phospho-Ulk1 (b), phospho-S6 (c), and p62 (d) in control, cortical tuber, and SEGA. e, Immunohistochemical staining for p62, phospho-S6, and synapsin in human control cortex and tissues from TSC patients, including tuber and SEGA (40× magnification). f, Quantifications of p62-positive cell counts from 20× magnification (mean ± SEM; n = 3–6; t test or ANOVA with Tukey's post hoc test; *p < 0.05; **p < 0.01; ***p < 0.001). Con, Control; Tuber, cortical tuber.

Figure 5.

Atg7KO mice display impaired autophagy and spontaneous recurrent seizures. a, Representative Western blots of lysates from the cortex, hippocampus, and cerebellum of wild-type and Atg7KO mice. b–d, Quantifications of LC3 ratio (b), p62 (c), and phospho-S6 (d) in cortex, hippocampus, and cerebellum in WT and Atg7KO mice (mean ± SEM; n = 3–6; t test; *p < 0.05; ***p < 0.001). e, Images of immunohistochemical staining of p62 in WT and Atg7KO mice in the hippocampus (CA1, CA3, dentate gyrus), cortex, and cerebellum acquired at 40× magnification. Ctx, Cortex; Hip, hippocampus; Crb, cerebellum.

Figure 6.

Atg7KO mice demonstrate spontaneous recurrent seizures and increased mortality a, Percentage of wild-type (circle) and Atg7KO (square) mice that displayed behavioral seizure activities by 20 weeks of age. b, Percentage of survival of wild-type and Atg7KO mice by 20 weeks of age. c, d, Representative electrographic recording in a WT mouse during a typical wake period (c) and an Atg7KO mouse during a spontaneous seizure episode (d).

Figure 7.

Atg7KO does not result in significant cell death. a, Representative H&E and cresyl violet staining of the hippocampus and cortex demonstrating no gross morphological abnormalities in Atg7KO mouse brains. b, TUNEL staining in the cortex, dentate gyrus (DG), and CA3 from age-matched 6- to 8-week-old WT and Atg7KO mice. Nuclease treatment serves as a positive control. Ctx, Cortex; Hip, hippocampus.