Abstracts

Rationale: Pten is a negative regulator of mTOR, and Pten loss-of-function (LOF) in neurons hyperactivates mTOR signaling and causes epilepsy in humans and animal models. Despite an association between Pten variants and cortical dysplasia in patients, the effects of Pten LOF on cortical neurons is relatively unexplored. Pten regulates both the mTORC1 and mTORC2 complexes, and previous research has demonstrated separate roles for the two complexes in neuronal function, synaptic transmission, and epilepsy. Genetic inhibition of either Raptor or Rictor allows the effects of Pten LOF to be studied through the two complexes separately. This approach has the potential to further elucidate specific mechanisms that lead to epilepsy induced by Pten LOF and other mTOR pathway genetic variants.

Methods: We induced Pten LOF, Pten and Raptor LOF, or Pten and Rictor LOF in neurons in the mouse neocortex in order to examine the effects of mTOR pathway hyperactivity on neuronal function and epilepsy. To assess neuronal physiology, whole cell patch clamp was conducted in cortical layer II/III in neurons expressing a fluorescent marker indicating LOF. A fluorescent Nissl stain was used to assess neuronal soma size. To assess seizure and interictal activity, mice were aged to adulthood and long-term EEG monitoring was conducted using skull surface electrodes.

Results: In neurons, Pten LOF caused a decrease in baseline membrane excitability accompanied by an increase in action potential firing at high levels of stimulation. Concurrent Raptor LOF rescued Pten LOF-induced membrane hypoexcitability to control levels and concurrent Rictor LOF partially rescued membrane hypoexcitability to an intermediate level. Similarly, neuronal soma size was increased in Pten LOF, rescued to control levels by Raptor LOF, and partially rescued by Rictor LOF. Pten LOF caused an increase in the number of spontaneous EPSCs onto the neuron, and this increase was not rescued by Raptor LOF or Rictor LOF. In live animals, long-term EEG monitoring showed epilepsy development in a subset of Pten LOF animals, and all Pten LOF animals showed substantial interictal epileptiform activity. We found that neither Raptor LOF nor Rictor LOF resulted in full rescue of epilepsy, with a subset of animals in each group developing epilepsy.

Conclusions: We find that epilepsy induced by Pten LOF is not mediated through solely mTORC1 or mTORC2, suggesting that either Pten LOF in neurons induces dysfunction through both pathways that can independently lead to epilepsy or that epilepsy caused by Pten LOF relies on some interaction between the two complexes. These results support previous evidence indicating that neuronal hypertrophy is not required for the development of epilepsy in Pten LOF and instead implicate Pten LOF-induced changes in synaptic function and connectivity.

Funding: Please list any funding that was received in support of this abstract.: NIH/NINDS K99/R00 NS087095; NIH/NINDS R01 NS110945.