Hyperactivity of mTORC1 or mTORC2-dependent signaling causes epilepsy downstream of somatic PTEN loss

Reviewer #1 (Public Review):

Hyperactivation of mTOR signaling causes epilepsy. It has long been assumed that this occurs through overactivation of mTORC1, since treatment with the mTORC1 inhibitor rapamycin suppresses seizures in multiple animal models. However, the recent finding that genetic inhibition of mTORC1 via Raptor deletion did not stop seizures while inhibition of mTORC2 did, challenged this view (Chen et al, Nat Med, 2019). In the present study, the authors tested whether mTORC1 or mTORC2 inhibition alone was sufficient to block the disease phenotypes in a model of somatic Pten loss-of-function (a negative regulator of mTOR). They found that inactivation of either mTORC1 or mTORC2 alone normalized brain pathology but did not prevent seizures, whereas dual inactivation of mTORC1 and mTORC2 prevented seizures. As the functions of mTORC1 versus mTORC2 in epilepsy remain unclear, this study provides important insight into the roles of mTORC1 and mTORC2 in epilepsy caused by Pten loss and adds to the emerging body of evidence supporting a role for both complexes in the disease development.

Strengths:
The animal models and the experimental design employed in this study allow for a direct comparison between the effects of mTORC1, mTORC2, and mTORC1/mTORC2 inactivation (i.e., same animal background, same strategy and timing of gene inactivation, same brain region, etc.). Additionally, the conclusions on brain epileptic activity are supported by analysis of multiple EEG parameters, including seizure frequencies, sharp wave discharges, interictal spiking, and total power analyses.

Weaknesses:
The sample size of the study is small and does not allow for the assessment of whether mTORC1 or mTORC2 inactivation reduces seizure frequency or incidence. This is a limitation of the study.

The authors describe that they inactivated mTORC1 and mTORC2 in a new model of somatic Pten loss-of-function in the cortex. This is slightly misleading since Cre expression was found both in the cortex and the underlying hippocampus, as shown in Figure 1. Throughout the manuscript, they provide supporting histological data from the cortex. However, since Pten loss-of-function in the hippocampus can lead to hippocampal overgrowth and seizures, data showing the impact of the genetic rescue in the hippocampus would further strengthen the claim that neither mTORC1 nor mTORC2 inactivation prevents seizures.

Some of the methods for the EEG seizure analysis are unclear. The authors describe that 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, whereas, for the Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF animals, at least 100 of the highest-amplitude traces were manually reviewed. Does this mean that not all flagged epochs were reviewed? This could potentially lead to missed seizures. Additionally, the inclusion of how many consecutive hours were recorded among the ~150 hours of recording per animal would help readers with the interpretation of the data.

Finally, it is surprising that mTORC2 inactivation completely rescued cortical thickness since such pathological phenotypes are thought to be conserved down the mTORC1 pathway. Additional comments on these findings in the Discussion would be interesting and useful to the readers.

Reviewer #2 (Public Review):

Summary:
The study by Cullen et al presents intriguing data regarding the contribution of mTOR complex 1 (mTORC1) versus mTORC2 or both in Pten-null-induced macrocephaly and epileptiform activity. The role of mTORC2 in mTORopathies, and in particular Pten loss-off-function (LOF)-induced pathology and seizures, is understudied and controversial. In addition, recent data provided evidence against the role of mTORC1 in PtenLOF-induced seizures. To address these controversies and the contribution of these mTOR complexes in PtenLOF-induced pathology and seizures, the authors injected a AAV9-Cre into the cortex of conditional single, double, and triple transgenic mice at postnatal day 0 to remove Pten, Pten+Raptor or Rictor, and Pten+raptor+rictor. Raptor and Rictor are essentially binding partners of mTORC1 and mTORC2, respectively. One major finding is that despite preventing mild macrocephaly and increased cell size, Raptor knockout (KO, decreased mTORC1 activity) did not prevent the occurrence of seizures and the rate of SWD event, and aggravated seizure duration. Similarly, Rictor KO (decreased mTORC2 activity) partially prevented mild macrocephaly and increased cell size but did not prevent the occurrence of seizures and did not affect seizure duration. However, Rictor KO reduced the rate of SWD events. Finally, the pathology and seizure/SWD activity were fully prevented in the double KO. These data suggest the contribution of both increased mTORC1 and mTORC2 in the pathology and epileptic activity of Pten LOF mice, emphasizing the importance of blocking both complexes for seizure treatment. Whether these data apply to other mTORopathies due to Tsc1, Tsc2, mTOR, AKT or other gene variants remains to be examined.

Strengths:
The strengths are as follows: 1) they address an important and controversial question that has clinical application, 2) the study uses a reliable and relatively easy method to KO specific genes in cortical neurons, based on AAV9 injections in pups. 2) they perform careful video-EEG analyses correlated with some aspects of cellular pathology.

Weaknesses:
The study has nevertheless a few weaknesses: 1) the conclusions are perhaps a bit overstated. The data do not show that increased mTORC1 or mTORC2 are sufficient to cause epilepsy. However the data clearly show that both increased mTORC1 and mTORC2 activity contribute to the pathology and seizure activity and as such are necessary for seizures to occur. 2) the data related to the EEG would benefit from having more mice. Adding more mice would have helped determine whether there was a decrease in seizure activity with the Rictor or Raptor KO. 3) it would have been interesting to examine the impact of mTORC2 and mTORC1 overexpression related to point #1 above.

Reviewer #3 (Public Review):

Summary: This study investigated the role of mTORC1 and 2 in a mouse model of developmental epilepsy which simulates epilepsy in cortical malformations. Given activation of genes such as PTEN activates TORC1, and this is considered to be excessive in cortical malformations, the authors asked whether inactivating mTORC1 and 2 would ameliorate the seizures and malformation in the mouse model. The work is highly significant because a new mouse model is used where Raptor and Rictor, which regulate mTORC1 and 2 respectively, were inactivated in one hemisphere of the cortex. The work is also significant because the deletion of both Raptor and Rictor improved the epilepsy and malformation. In the mouse model, the seizures were generalized or there were spike-wave discharges (SWD). They also examined the interictal EEG. The malformation was manifested by increased cortical thickness and soma size.

Strengths: The presentation and writing are strong. The quality of data is strong. The data support the conclusions for the most part. The results are significant: Generalized seizures and SWDs were reduced when both Torc1 and 2 were inactivated but not when one was inactivated.

Weaknesses: One of the limitations is that it is not clear whether the area of cortex where Raptor or Rictor were affected was the same in each animal. Also, it is not clear which cortical cells were measured for soma size. Another limitation is that the hippocampus was affected as well as the cortex. One does not know the role of cortex vs. hippocampus. Any discussion about that would be good to add. It would also be useful to know if Raptor and Rictor are in glia, blood vessels, etc.

Author Response

eLife assessment

This useful study addresses epilepsy caused by the loss of a molecule called Pten, resulting in hyperactivity of the mTOR pathway. The findings suggest that inhibiting two molecules called mTORC1 and mTORC2 can reduce epilepsy symptoms but there is much less effect when inhibited separately. The evidence supporting the conclusions is currently incomplete, but could be strengthened after additional experiments.

We thank the editors for this assessment and the reviewers for their comments. We will consider each of the recommendations we received and revise the manuscript accordingly.

Reviewer #1 (Public Review):

Hyperactivation of mTOR signaling causes epilepsy. It has long been assumed that this occurs through overactivation of mTORC1, since treatment with the mTORC1 inhibitor rapamycin suppresses seizures in multiple animal models. However, the recent finding that genetic inhibition of mTORC1 via Raptor deletion did not stop seizures while inhibition of mTORC2 did, challenged this view (Chen et al, Nat Med, 2019). In the present study, the authors tested whether mTORC1 or mTORC2 inhibition alone was sufficient to block the disease phenotypes in a model of somatic Pten loss-of-function (a negative regulator of mTOR). They found that inactivation of either mTORC1 or mTORC2 alone normalized brain pathology but did not prevent seizures, whereas dual inactivation of mTORC1 and mTORC2 prevented seizures. As the functions of mTORC1 versus mTORC2 in epilepsy remain unclear, this study provides important insight into the roles of mTORC1 and mTORC2 in epilepsy caused by Pten loss and adds to the emerging body of evidence supporting a role for both complexes in the disease development.

Strengths:

The animal models and the experimental design employed in this study allow for a direct comparison between the effects of mTORC1, mTORC2, and mTORC1/mTORC2 inactivation (i.e., same animal background, same strategy and timing of gene inactivation, same brain region, etc.). Additionally, the conclusions on brain epileptic activity are supported by analysis of multiple EEG parameters, including seizure frequencies, sharp wave discharges, interictal spiking, and total power analyses.

Weaknesses:

1. The sample size of the study is small and does not allow for the assessment of whether mTORC1 or mTORC2 inactivation reduces seizure frequency or incidence. This is a limitation of the study.

We agree that this is a minor limitation of the present study, however, for several reasons we decided not to pursue this question by increasing the number of animals. First, we performed a power analysis of the existing data. This analysis showed that we would need to use 89 animals per group to detect a significant difference (0.8 Power, p= 0.05, Mann-Whitney test) in the frequency of generalized seizures in the Pten-Raptor group and 31 animals per group in the Pten-Rictor group versus Pten alone. It is simply not feasible to perform EEG monitoring on this many animals. Second, even if we did do enough experiments to detect a reduction in seizure frequency, it is clear that neither Raptor nor Rictor deletion provides the kind normalization in brain activity that we seek in a targeted treatment. Both Pten-Raptor and Pten-Rictor animals still have very frequent spike-wave events (Fig. 3D) and highly abnormal interictal EEGs (Fig. 4), suggesting that even if generalized seizures were reduced, epileptic brain activity persists. This is in contrast to the triple KO animals, which have no increase in SWD above control level and very normal interictal EEG.

1. The authors describe that they inactivated mTORC1 and mTORC2 in a new model of somatic Pten loss-of-function in the cortex. This is slightly misleading since Cre expression was found both in the cortex and the underlying hippocampus, as shown in Figure 1. Throughout the manuscript, they provide supporting histological data from the cortex. However, since Pten loss-of-function in the hippocampus can lead to hippocampal overgrowth and seizures, data showing the impact of the genetic rescue in the hippocampus would further strengthen the claim that neither mTORC1 nor mTORC2 inactivation prevents seizures.

Thank you for pointing out this issue. Cre expression was observed in both the cortex and the dorsal hippocampus in most animals, and we agree that differences in cortical versus hippocampal mTOR signaling could have differential contributions to epilepsy. We focused our studies on the cortex because spike-and-wave discharge, the most frequent and fully penetrant EEG phenotype in our model, is associated with cortical dysfunction. We had also performed a preliminary analysis of the hippocampal Cre expression, which suggested that Cre expression in the hippocampus did not affect generalized seizure occurrence. We plan to include data on Cre expression in the hippocampus in the revised version of the manuscript.

1. Some of the methods for the EEG seizure analysis are unclear. The authors describe that 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, whereas, for the Pten LOF, Pten-Raptor LOF, and Pten-Rictor LOF animals, at least 100 of the highest-amplitude traces were manually reviewed. Does this mean that not all flagged epochs were reviewed? This could potentially lead to missed seizures.

We reviewed at least 48 hours of data from each animal manually. All seizures that were identified during manual review were also identified by the automated detection program. It is possible but unlikely that there are missed seizures in the remaining data.

1. Additionally, the inclusion of how many consecutive hours were recorded among the ~150 hours of recording per animal would help readers with the interpretation of the data.

Thank you for this recommendation. We plan to include a table with more information about the EEG recordings in the revised version of the manuscript. The number of consecutive hours recorded varied because the wireless system depends on battery life, which was inconsistent, but each animal was recorded for at least 48 consecutive hours on at least two occasions.

1. Finally, it is surprising that mTORC2 inactivation completely rescued cortical thickness since such pathological phenotypes are thought to be conserved down the mTORC1 pathway. Additional comments on these findings in the Discussion would be interesting and useful to the readers.

Soma size was increased 120% by Pten inactivation and partially normalized to a 60% increase from Controls by mTORC2 inactivation (Fig. 2C). We and others have previously shown that mTORC2 inactivation in neurons reduces both soma size and dendritic outgrowth (PMIDs: 36526374, 32125271, 23569215). Thus, we do not find it completely surprising that mTORC2 inactivation reduces the cortical thickness increase caused by Pten loss. There may still be a slight increase in cortical thickness in Pten-Rictor animals, but it is statistically indistinguishable from Controls. We will elaborate on this in our revised submission.

Reviewer #2 (Public Review):

Summary:

The study by Cullen et al presents intriguing data regarding the contribution of mTOR complex 1 (mTORC1) versus mTORC2 or both in Pten-null-induced macrocephaly and epileptiform activity. The role of mTORC2 in mTORopathies, and in particular Pten loss-off-function (LOF)-induced pathology and seizures, is understudied and controversial. In addition, recent data provided evidence against the role of mTORC1 in PtenLOF-induced seizures. To address these controversies and the contribution of these mTOR complexes in PtenLOF-induced pathology and seizures, the authors injected a AAV9-Cre into the cortex of conditional single, double, and triple transgenic mice at postnatal day 0 to remove Pten, Pten+Raptor or Rictor, and Pten+raptor+rictor. Raptor and Rictor are essentially binding partners of mTORC1 and mTORC2, respectively. One major finding is that despite preventing mild macrocephaly and increased cell size, Raptor knockout (KO, decreased mTORC1 activity) did not prevent the occurrence of seizures and the rate of SWD event, and aggravated seizure duration. Similarly, Rictor KO (decreased mTORC2 activity) partially prevented mild macrocephaly and increased cell size but did not prevent the occurrence of seizures and did not affect seizure duration. However, Rictor KO reduced the rate of SWD events. Finally, the pathology and seizure/SWD activity were fully prevented in the double KO. These data suggest the contribution of both increased mTORC1 and mTORC2 in the pathology and epileptic activity of Pten LOF mice, emphasizing the importance of blocking both complexes for seizure treatment. Whether these data apply to other mTORopathies due to Tsc1, Tsc2, mTOR, AKT or other gene variants remains to be examined.

Strengths:

The strengths are as follows: 1) they address an important and controversial question that has clinical application, 2) the study uses a reliable and relatively easy method to KO specific genes in cortical neurons, based on AAV9 injections in pups. 2) they perform careful video-EEG analyses correlated with some aspects of cellular pathology.

Weaknesses:

The study has nevertheless a few weaknesses: 1) the conclusions are perhaps a bit overstated. The data do not show that increased mTORC1 or mTORC2 are sufficient to cause epilepsy. However the data clearly show that both increased mTORC1 and mTORC2 activity contribute to the pathology and seizure activity and as such are necessary for seizures to occur.

We agree that our findings do not directly show that either mTORC1 or mTORC2 hyperactivity are sufficient to cause seizures, as we do not individually hyperactivate each complex in the absence of any other manipulation. We interpreted our findings in this model as suggesting that either is sufficient based on the result that there is no epileptic activity when both are inactivated, and thus assume that there is not a third, mTOR-independent, mechanism that is contributing to epilepsy in Pten, Pten-Raptor, and Pten-Rictor animals. In addition, the histological data show that Raptor and Rictor loss each normalize activity through mTORC1 and mTORC2 respectively, suggesting that one in the absence of the other is sufficient. However, we agree that there could be other potential mTOR-independent pathways downstream of Pten loss that contribute to epilepsy. We will revise the manuscript to reflect this.

1. the data related to the EEG would benefit from having more mice. Adding more mice would have helped determine whether there was a decrease in seizure activity with the Rictor or Raptor KO.

Please see response to Reviewer 1’s first Weakness.

1. it would have been interesting to examine the impact of mTORC2 and mTORC1 overexpression related to point #1 above.

We are not sure that overexpression of individual components of mTORC1 or mTORC2 would result in their hyperactivation or lead to increases in downstream signaling. We believe that cleanly and directly hyperactivating mTORC1 or especially mTORC2 in vivo without affecting the other complex or other potential interacting pathways is a difficult task. Previous studies have used mTOR gain-of-function mutations as a means to selectively activate mTORC1 or pharmacological agents to selectively activate mTORC2, but it not clear to us that the former does not affect mTORC2 activity as well, or that the latter achieves activation of mTORC2 targets other than p-Akt 473, or that it is truly selective. We agree that these would be key experiments to further test the sufficiency hypothesis, but that the amount of work that would be required to perform them is more that what we can do in this Short Report.

Reviewer #3 (Public Review):

Summary: This study investigated the role of mTORC1 and 2 in a mouse model of developmental epilepsy which simulates epilepsy in cortical malformations. Given activation of genes such as PTEN activates TORC1, and this is considered to be excessive in cortical malformations, the authors asked whether inactivating mTORC1 and 2 would ameliorate the seizures and malformation in the mouse model. The work is highly significant because a new mouse model is used where Raptor and Rictor, which regulate mTORC1 and 2 respectively, were inactivated in one hemisphere of the cortex. The work is also significant because the deletion of both Raptor and Rictor improved the epilepsy and malformation. In the mouse model, the seizures were generalized or there were spike-wave discharges (SWD). They also examined the interictal EEG. The malformation was manifested by increased cortical thickness and soma size.

Strengths: The presentation and writing are strong. The quality of data is strong. The data support the conclusions for the most part. The results are significant: Generalized seizures and SWDs were reduced when both Torc1 and 2 were inactivated but not when one was inactivated.

Weaknesses: One of the limitations is that it is not clear whether the area of cortex where Raptor or Rictor were affected was the same in each animal.

We plan to include data further describing the location of knockout in each animal (in both the hippocampus and cortex) in the revised version of the paper. Initial analyses indicated that the affected area did not differ between groups.

Also, it is not clear which cortical cells were measured for soma size.

In the Methods it says “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.” Earlier under “Histology and imaging” it says “Three sections per animal at approximately Bregma -1.6, -2,1, and -2.6 were used.”

Another limitation is that the hippocampus was affected as well as the cortex. One does not know the role of cortex vs. hippocampus. Any discussion about that would be good to add.

See response to Reviewer 1’s second Weakness.

It would also be useful to know if Raptor and Rictor are in glia, blood vessels, etc.

Raptor and Rictor are thought to be ubiquitously active in mammalian cells including glia and endothelial cells. Previous studies have shown that mTOR manipulation can affect astrocyte function and blood vessel organization, however, our study induced gene knockout using an AAV that expressed Cre under control of the hSyn promoter, which has previously been shown to be selective for neurons. Manual assessment of Cre expression compared with DAPI, NeuN, and GFAP stains suggested that only neurons were affected.