Introduction

Genetic disruptions that hyperactivate mTOR activity, termed ‘mTORopathies’, cause macrocephaly, intractable childhood epilepsies, and behavioral presentations including autism spectrum disorders [1-4]. The mTOR kinase can function in two complexes, mTORC1 and mTORC2, each with unique downstream targets and upstream activators [5]. A unifying feature of mTORopathies is hyperactivation of mTORC1, which has led to the hypothesis that this complex and its effectors mediate disease phenotypes [6]. A subset of mTORopathies however, (e.g. those caused by variants in PTEN, PIC3CA, and AKT), hyperactivate both mTORC1 and mTORC2 [7,8], and mTORC2 hyperactivity has also been reported in human TLE [9]. It is unclear whether mTORC2 hyperactivity contributes to, or can cause, disease phenotypes such as epilepsy.

PTEN LOF in neurons induces soma hypertrophy, increased dendritic branching, and synaptic hyperconnectivity [10,11]. These phenotypes resemble those observed in models of mTORC1-specific hyperactivation [12], and can be prevented with either the mTORC1 inhibitor rapamycin or loss of the mTORC1-specific protein Raptor [13]. Rapamycin also rescues epilepsy and mortality associated with Pten loss, even after epilepsy has been established, suggesting that mTORC1 hyperactivity underlies these phenotypes [14-16]. Thus, there is strong evidence that hyperactivation of mTORC1 downstream of PTEN disruption causes the macrocephaly, epilepsy, early mortality, and synaptic dysregulation observed in humans and model organisms [17].

There is also evidence against the mTORC1-centric hypothesis. Rapamycin may affect mTORC2 activity at high doses or over long periods of time [18,17], and mTORC2 regulates synapse function, neuronal size, and cytoskeletal organization independent of mTORC1 [19,20]. In a mouse model in which Pten was inactivated in forebrain neurons in early adulthood, mTORC2 inhibition, but not mTORC1 inhibition, rescued seizures and behavioral abnormalities. In this model, epilepsy was dissociated from macrocephaly, which was rescued by mTORC1 inhibition but not mTORC2 inhibition [21]. However, mTORC2 inactivation did not normalize spontaneous seizures or seizure susceptibility in a model of Pten loss in dentate granule neurons [22]. Thus, there is conflicting evidence about whether mTORC1 or mTORC2 hyperactivity causes epilepsy downstream of Pten loss.

To address this, we suppressed mTORC1 and mTORC2 activity alongside PTEN LOF by inducing simultaneous deletion of Raptor or Rictor, whose protein products are unique and essential components of mTORC1 and mTORC2, respectively, in a model of somatic PTEN LOF in the cortex. In this model, epilepsy could be rescued by concurrent mTORC1 and mTORC2 inactivation, but persisted when either gene remained intact, suggesting that hyperactivity of either complex can lead to neuronal hyperexcitability and epilepsy.

Results

Generation of a developmental brain somatic mosaic model of PTEN LOF

To model the epileptogenic somatic mutations often observed in mTORopathies [23,24], we injected an AAV9 virus expressing GFP and Cre under control of the hSyn promoter into one hemisphere of the cortex of Pten^(fl/fl), Pten^(fl/fl)-Raptor^(fl/fl), Pten^(fl/fl)-Rictor^(fl/fl), and Pten^(fl/fl)-Raptor^(fl/fl)-Rictor^(fl/fl) mice at P0 (Fig. 1A). Green fluorescent protein (GFP) was expressed in neurons throughout the cortical layers in all experimental animals, but largely confined to one cortical hemisphere and the underlying hippocampus (Fig. 1B). The percentage of cells that were GFP+ was similar across groups, although there was a decrease in cell density in the Pten LOF and Pten-Rictor LOF groups [25] (Fig. 1C).

Figure 1.

We analyzed the impact of Pten, Raptor, and Rictor LOF on mTORC1 and mTORC2 activity in the affected hemisphere using quantitative immunohistochemistry. Phospho-S6 levels, which report mTORC1 activity through S6K1, were increased from Controls in Pten LOF and Pten-Rictor LOF brains, but rescued in Pten-Raptor LOF brains (Fig. 1D₁). Phospho-Akt (S473) levels, which report mTORC2 activity, were increased in Pten LOF and Pten-Raptor LOF but normalized in Pten-Rictor LOF brains (Fig. 1D₂). Levels of both pS6 and pAkt were not different from Controls in Pten-Raptor-Rictor LOF brains. These results indicate that Pten LOF hyperactivates both mTORC1 and mTORC2 in this model, and that these increases in activity are independently rescued by mTORC1 and mTORC2 inactivation.

Raptor and/or Rictor LOF attenuate Pten LOF-induced macrocephaly

Macrocephaly and neuronal hypertrophy are well-characterized sequelae of deficient PTEN signaling in patients and mouse models [26,27]. In mouse models of Pten LOF, these phenotypes can be rescued by rapamycin treatment or Raptor LOF [21,28,17,13]. Here, cortical thickness was increased in Pten LOF and rescued to Control levels by Raptor LOF, Rictor LOF, or combined Raptor-Rictor LOF (Fig. 2A-B). In individual neurons Pten LOF caused a ≈100% increase in mean soma size versus Control. Pten-Rictor LOF significantly reduced soma size from Pten LOF levels, but still showed a 60% increase over Controls. Soma size in Pten-Raptor LOF and Pten-Raptor-Rictor LOF neurons did not significantly differ from Controls (Fig. 2C).

Figure 2.

Concurrent mTORC1/2 inactivation, but neither alone, rescues epilepsy and interictal EEG abnormalities in focal Pten LOF

Next, we measured epileptic brain activity with video-EEG to determine the ability of mTORC1 or mTORC2 activity to rescue this key feature of mTORopathies, Generalized seizures (GS) were observed in 0/9 Control, 4/7 Pten LOF, 2/6 Pten-Raptor LOF, 2/7 Pten-Rictor LOF, and 0/6 Pten-Raptor-Rictor LOF animals. GS events were longer-lasting in Pten-Raptor LOF animals than in Pten LOF or Pten-Rictor LOF animals (Fig. 3A). GS did not appear to be correlated with mTOR pathway activity (Supplementary Figure 2).

Figure 3.

The most striking and consistent type of epileptic brain activity in the Pten LOF animals was frequent 5-7 Hz spike trains lasting 3 seconds or more, which fit previous characterizations of spike- and-wave discharges (SWDs) in rodents [29]. SWDs were observed in all Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF animals, and in 3/9 Control and 3/6 Pten-Raptor-Rictor LOF animals. The frequency of SWDs in Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF animals was significantly higher than Controls, but Pten-Raptor-Rictor LOF completely blocked this increase. Pten-Rictor LOF animals had a lower frequency of SWD events than Pten LOF animals (Fig. 3B), indicating a partial rescue. Taken together, these data indicate inactivating mTORC1 provides no protection against Pten loss-induced epilepsy, and may even exacerbate it. mTORC2 inactivation provides some protection, but only concurrent mTORC1 and mTORC2 inactivation can prevent it.

In addition to epileptic activity, we also found that Pten LOF caused obvious alterations in features of the interictal EEG. To quantify these changes, we measured EEG coastline, absolute mean amplitude, and power spectra of the EEG. Coastline, mean amplitude, and total power were all significantly increased by Pten LOF, and these changes were not significantly reduced by Pten-Raptor or Pten-Rictor LOF. Pten-Raptor-Rictor LOF, however, reduced these increases to Control levels. Correspondingly, EEG power was increased in Pten LOF animals (Fig. 4A; Table 1A). This increase was normalized by Pten-Raptor-Rictor LOF, but not by Pten-Raptor LOF or Pten-Rictor LOF. Pten-Raptor-Rictor LOF did not fully rescue a rightward shift in normalized EEG band power (Fig. 4B; Table 1B).

Table 1.

Figure 4.

Discussion

Convergent discoveries have positioned mTORopathies as prime candidates for a precision medicine approach [30,31]. There has been some clinical success with rapamycin analogues [32], but preclinical animal studies have suggested ways to further improve the selectivity and efficacy of the targets [12]. For the subset of mTORopathies in which mTORC1 but not mTORC2 signaling is hyperactive, selective inhibition of mTORC1 and its downstream effectors rescues many animal phenotypes, including seizures [33,34]. Studies using rapamycin as an mTORC1 inhibitor also came to this conclusion in Pten LOF models, but potential inhibition of mTORC2 and the finding that genetic inhibition of mTORC1 via Raptor deletion did not stop seizures, whereas inhibition of mTORC2 did [21], challenged this view. Here, we tested whether mTORC1 or mTORC2 inhibition alone were sufficient to block disease phenotypes in a model of somatic Pten LOF. Neither was, which agrees with previous findings that seizures persist in both Pten-Raptor LOF and Pten-Rictor LOF animals in separate model systems [21,22]. This data could be interpreted as suggesting that epilepsy downstream of Pten LOF can proceed via mTORC-independent mechanisms, such as elevated ß-catenin or its protein phosphatase activity [25,35]. However, we found that simultaneous mTORC1 and mTORC2 inhibition corrected all of the pathological features of our model except for a rightward shift in relative EEG power (Fig. 4B). This suggests that epilepsy can be caused by hyperactivity of either mTORC1 or mTORC2-dependent signaling downstream of Pten LOF, but not alternative pathways. Future studies should determine whether this is also true for other gene variants that hyperactivate both mTORC1 and mTORC2, as well in other models that have fully penetrant generalized seizures.

Epilepsy in Pten-Rictor LOF mice likely emerges via mechanisms similar to those in mTOR pathway variants such as TSC that hyperactivate mTORC1 but not mTORC2, although additional effects of mTORC2 inactivation may contribute [22]. Epilepsy in Pten-Raptor LOF occurs in the absence of macrocephaly, a hallmark of mTORC1 hyperactivity, possibly due to mTORC2 hyperactivity’s effect on synaptic transmission [20]. Roles for both complexes in emergent neural network function are corroborated by findings that behavior can be altered by either mTORC1 or mTORC2 inactivation [36]. Rapamycin derivatives are somewhat selective for mTORC1 [37,38] and are currently being tested in the clinic to treat mTORopathies and PTEN-related disorders [18]. Even more selective targeting of mTORC1 than can be achieved through rapamycin and its derivatives has been proposed as a way to effectively treat disease with fewer side effects [39]. Our data suggest that this may only be true for mTORopathies that only hyperactive mTORC1. For others, including PTEN, PIK3CA, and AKT, dual inhibitors or inhibitors of PI3K, which are being tested preclinically [40-42], are likely to be required.

Materials and Methods

Animals

Raptor (Jackson Labs #013188) and Rictor (Jackson Labs #020649) homozygous floxed mice were crossed to a Pten homozygous floxed line ([43] Jackson Labs #006440). Experimental animals were the offspring of two animals homozygous floxed for Pten and heterozygous floxed for Raptor and/or Rictor. Intracranial viral injections were done at P0 or P1. Animals that were homozygous for the floxed Pten allele and either homozygous or wild-type at the Raptor and/or Rictor allele were injected with an AAV9 viral vector expressing an eGFP-Cre fusion driven by the hSyn promoter (Addgene #105540). Control animals were of the same genotype but were injected with an AAV9 virus expressing eGFP under control of the hSyn promoter, but not Cre (Addgene #105539). Pup injections were conducted with hypothermic anesthesia. A Nanoject was used to deliver 100 nL of Cre or control virus to three sites spanning the left hemisphere of the cortex. Pups were quickly rewarmed and returned to their home cage. Additional control animals with no floxed genes on a C57B6/J background (Jackson Labs #000644) were injected with the eGFP-Cre expressing AAV9 to ensure that Cre expression did not have effects independent of the Pten^fl/fl genotype (Supplementary Figure 1).

Surgeries and EEG Recordings

Animals were aged to six weeks and then implanted with a wireless EEG monitoring headcap. Animals were anesthetized with 4% isofluorane and arranged on a stereotaxic surgical apparatus with ear bars. Anesthesia was maintained with 1.5-2.5% isofluorane. The skull surface was exposed and holes were made with a 23g needle at six locations on the skull surface. 3/32” screws (Antrin Miniature Specialties) were inserted into the holes. Screws were secured with VetBond then covered with surgical cement. The screws were attached to a six-pin Millimax strip which was secured to the skull with additional cement. Animals were administered 5 mg/kg ketoprofen post-surgery and allowed to recover for at least 5 days before EEG recording. Surgical protocol was based on guidance provided by Pinnacle Technologies (https://www.pinnaclet.com).

After recovery from surgery, animals were fitted with wireless three-channel EEG preamplifiers (Pinnacle Technologies) and EEG signal was recorded at 256 Hz with simultaneous video recording. EEG recording session length was dependent on battery life. A total of at least 150 hours of EEG data was collected for each experimental animal.

Histology and imaging

EEG-implanted animals were euthanized by isofluorane overdose followed by transcardiac perfusion with 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde for 24 hours and then transferred to 30% sucrose for at least 48 hours. 40 μm coronal slices were cut and preserved in a cryoprotectant solution (50% PBS, 30% ethylene glycol, 20% glycerol). Prior to staining, slices were washed three times with PBS. Eight cross-sectional unstained slices were mounted with DAPI Fluoromount-G (SouthernBiotech) and used to assess cortical thickness, GFP expression, and cell density. For other analyses, three sections per animal at approximately Bregma -1.6, -2,1, and -2.6 were used. A fluorescent Nissl stain (Invitrogen N21482, 1:50) was used to assess soma size.

After washing, slices were placed in blocking solution (10% normal goat serum, 0.1% Triton X-100, and PBS) for 1 hour. Slices were incubated in the following primary antibodies for 3 hours at room temperature: phospho-S6 Ribosomal Protein Ser240/244 (rabbit monoclonal, 1:1000 dilution, Cell Signaling Technology, catalog #5364, RRID: AB_10694233), phospho-AKT Ser473 (rabbit monoclonal, 1:1000 dilution, Cell Signaling Technology, catalog #4060), and NeuN (guinea pig polyclonal, 1:1000 dilution, Synaptic Systems, catalog #266004). Following primary antibody application, slices were washed three times in PBS and the incubated in AlexaFluor secondary antibodies goat anti-guinea pig 647 and goat anti-rabbit 594 (Invitrogen) for 1 hour at room temperature.

Slides stained for Nissl substance, NeuN/pS6, and NeuN/pAkt were imaged at 10x (2048×2048) with 5 μm z-step on a Nikon C2 confocal microscope (UVM Microscopy Imaging Core). One image of the left (GFP-expressing) cortical hemisphere in each of three region-matched slices per animal was collected for analysis. Widefield images (2560×2160) of unstained DAPI-mounted cross-sectional slices were taken with a Zyla sCMOS camera (Andor) mounted on an upright microscope (IX73; Olympus) at 5x and 20x resolution. Pixel width was manually calculated using an 0.1 mm hemocytometer.

EEG analysis

EEG files were converted from Pinnacle’s proprietary format to European Data Format (EDF) files and imported to Matlab. A filtering program was used to flag traces in which seizures were likely to be occurring based on amplitude and line length between data points. For Control and Pten-Raptor-Rictor LOF animals, all 10 second epochs in which signal amplitude exceeded 400 μV at two time points at least 1 second apart were manually reviewed by a rater blinded to genotype. This method was not suitable for the Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF animals because of the density of high-amplitude interictal activity they displayed. For these animals, at least 100 of the highest-amplitude traces were manually reviewed and then traces with persistent abnormally low amplitude, often indicating postictal suppression, were reviewed as well. Flagged traces were displayed for a rater to mark the beginning and end of each seizure. Spike-and-wave discharge events were manually marked in 8 evenly spaced one-hour epochs in each of two 24-hour recording sessions per animal and verified on video not to be caused by movement such as chewing or scratching.

Baseline EEG measures were taken from a representative sample of EEG files for each animal. 692 evenly spaced five-second epochs were sampled over 24 hours, repeated for two recording sessions per animal. Line length was defined as the sum of the linear distances between adjacent data points during the five-second analysis epoch. Mean amplitude was defined as the mean of the absolute value of data points in the five-second analysis epoch. Power spectral density was calculated with the pwelch function in Matlab. All three EEG channels were analyzed and the mean of all channels was used for statistical analysis.

Image Analysis

Image analysis was conducted using ImageJ/Fiji. Soma size was measured by dividing Nissl stain images into a 10 mm² grid. The somas of all GFP-expressing cells fully within three randomly selected grid squares in Layer II/III were manually traced. pS6 and pAkt expression were measured by drawing 200 μm wide columns spanning all cortical layers. Background was subtracted from images with a 30 μm rolling ball algorithm (Fiji/ImageJ). The mean pixel value of the column was recorded and values were averaged by animal.

Statistical analysis

Prism 9 (GraphPad Prism) was used to conduct statistical analyses and create graphs. Group differences were assessed using one-way ANOVA with Tukey post-hoc analysis in GraphPad9, except where noted.

Acknowledgements

Research reported in this publication was supported by NINDS grant R01NS110945 (M.C.W.), as well as P20GM135007, Core C: Customized Physiology and Imaging Core. Images created with BioRender. We thank Caitlynn Barrows, Elise Prehoda, and Willie Tobin for early experiments helping create the mouse model.