Characterization of the focal cortical Pten LOF model.

A) Experimental timeline showing induction of hSyn-Cre-GFP or Control hSyn-GFP AAV at P0 and EEG recording in adulthood. B) Representative images of GFP expression in a Control mouse brain, demonstrating expression predominantly in one hemisphere of the cortex. C1) Quantification of lesion severity and neuron density in Cre-expressing animals. There were fewer Cre-expressing neurons per unit area in the Pten LOF and Pten-Rictor LOF groups, which was at least partially attributable to a decrease in cell density in these groups. No significant differences in Cre expression remained when Cre expression was calculated based on cell density rather than area. C2) Representative images of DAPI and Cre fluorescence in the cortex of Pten LOF and Pten-Raptor LOF animals. Cell density and Cre density in Pten-Raptor LOF animals was indistinguishable from Controls. D1) Phospho-S6, a marker of mTORC1 activity, was increased by Pten LOF and reduced to control levels by concurrent Raptor LOF. Phospho-S6 was also increased from Control levels in Pten-Rictor LOF, indicating that mTORC1 hyperactivity was not normalized by Rictor LOF. Combined Raptor/Rictor LOF also normalized phospho-S6 expression. D2) phospho-Akt, a marker of mTORC2 activity, was increased in Pten LOF and normalized by Rictor LOF, but not by Raptor LOF. Combined Raptor/Rictor LOF also normalized phospho-Akt expression. Error bars show mean ± s.e.m. ns indicates p>0.05, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 as assessed by one-way ANOVA with Tukey multiple comparisons correction. Diagram created with BioRender.com.

mTORC1 inhibition or dual mTORC1/2 inhibition rescues, and mTORC2 inhibition partially rescues, abnormalities in cortical morphology induced by focal Pten LOF.

A) Example images showing fluorescent Nissl stain (red) and GFP expression (green) in cortical neurons. The top row shows the cortical thickness in all 5 groups and the bottom row shows zoomed in images depicting the differences in soma size across groups. B) The mean cortical thickness was increased in Pten LOF throughout the cortex. Pten-Raptor, Pten-Rictor, and Pten-Raptor-Rictor LOF cortical thickness did not differ significantly from Controls. C) The mean soma size was strongly increased in Pten LOF and to a smaller extent in Pten-Rictor LOF. Pten-Raptor LOF and Pten-Raptor-Rictor LOF groups did not differ significantly from Controls. Error bars show mean ± s.e.m. ns indicates p>0.05, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001 as assessed by one-way ANOVA with Tukey multiple comparisons correction.

Combined mTORC1 and mTORC2 inactivation, but neither alone, rescues epilepsy in the focal Pten LOF model.

Spontaneous seizures and interictal spike activity were assessed in mice expressing Pten LOF, Pten-Raptor LOF, Pten-Rictor LOF, Pten-Raptor-Rictor LOF, and Controls. A) Representative traces of GS in a subset of animals in Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF groups. B) Spectrograms (top) and traces depicting example SWD event trains. C) Summary data showing the number of GS per day and GS length. GS events were significantly longer in the Pten-Raptor LOF group than the other groups. In the Pten LOF group, 19% of GS events (9/48) exceeded 45 seconds in length, and these events were observed in 2/4 Pten LOF GS+ animals. 79% of GS events (15/19) in the Pten-Raptor LOF group exceeded 45 seconds, and these events were observed in 2/2 GS+ Pten-Raptor LOF animals. 1/11 GS events in the Pten-Rictor LOF group exceeded this threshold. D. Summary data showing the SWD rate and length in all animals. Error bars show mean ± s.e.m. ns indicates p>0.05, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 as assessed by one-way ANOVA with Tukey multiple comparisons correction.

EEG power

Combined mTORC1 and mTORC2 inactivation, but neither alone, rescues Pten LOF-induced abnormalities in the interictal EEG.

A) Examples of typical EEG traces for each genotype. In EEG epochs that were not characterized as GTCS or SWD events, Pten LOF animals had higher levels of EEG activity as quantified by EEG line length, absolute mean amplitude, and total power. These changes were not significantly decreased by Raptor LOF and Rictor LOF but were normalized by combined Raptor/Rictor LOF. B) Total EEG power was increased by Pten LOF and attenuated but not normalized by either Raptor or Rictor LOF. Relative power was decreased in delta and increased higher frequencies by Pten LOF. Pten-Raptor LOF, Pten-Rictor LOF, and Pten-Raptor-Rictor LOF animals all showed a milder rightward shift of EEG power. C) Line length, mean amplitude, and power are increased in Pten LOF and normalized by Pten-Raptor-Rictor LOF. Error bars show mean ± s.e.m. ns indicates p>0.05, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 as assessed by one-way ANOVA with Tukey multiple comparisons correction. Two-way ANOVA p-values for EEG power are reported in Tables 1 and 2.

Cre virus exposure does not significantly impact cortical morphology or baseline EEG.

C57B6/J mice with no floxed genes were injected with the Cre virus as an additional control group. These mice did not have GS or SWDs. They were also not different from Ptenfl/fl injected with the Control virus, except for significantly different brain weights. The brain weight/body weight ratio did not differ between the groups.

Focal cortical Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF cause a spectrum of outcomes.

A subset of animals in the Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF groups displayed spontaneous epileptiform activity, but not generalized seizures. When compared side by side, animals within each genotype that did and did not display generalized seizures showed similar mTOR pathway activity levels and soma sizes. Survival plot shows survival of animals in the study by genotype. Some animals in the Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF groups were found dead during the study, but no deaths were observed in Control or Pten-Raptor-Rictor LOF groups. Mortality often occurred prior to EEG recordings, so we could not ascertain whether early mortality was associated with generalized seizures.