UC Berkeley

The mechanistic target of rapamycin (mTOR) is a kinase found in two multi-protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). These complexes are integral parts of an evolutionarily conserved signaling pathway known as the mTOR pathway. mTORC1 and mTORC2 are controlled by an array of intra and extracellular stimuli via different upstream regulators. The two complexes are known to exert distinct functions by phosphorylating downstream targets and ultimately orchestrate cell growth and metabolism. Aberrant mTOR activity is associated with numerous diseases, with particularly profound impact on the nervous system. Specifically, mutations in genes encoding for mTOR regulators result in a collection of neurodevelopmental disorders known as mTORopathies.Tuberous Sclerosis Complex (TSC) is one of the most well characterized mTORopathies. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode proteins that negatively regulate mTORC1 signaling. Current therapeutic strategies focus on rapamycin and its analogs that are inhibitors of mTORC1. However, several studies have shown that chronic rapamycin inhibits both mTORC1 and mTORC2 in a cell-type specific manner, raising the possibility that mTORC2 suppression might also exert therapeutic benefits in TSC. This idea has been corroborated by some studies showing that mTORC2 is involved in cellular processes that are altered in TSC, such as myelination and mGluR-dependent synaptic long-term depression. Most recently a study showed that mTORC2 suppression can provide therapeutic benefits in other mTORopathies. It is currently unknown which mTOR complex is most relevant for TSC-related brain phenotypes. To model TSC we used in vitro systems of primary hippocampal cultures where we examined postnatal loss of Tsc1 and we also used the Emx1-Cre mouse line to conditionally delete Tsc1 embryonically from forebrain excitatory neurons. To investigate which mTOR complex is responsible for TSC neurologic manifestations we used genetic strategies to target Raptor and Rictor and selectively reduce mTORC1 or mTORC2 activity respectively. Interestingly, our study revealed that the two complexes regulate each other’s activity and loss of either Raptor or Rictor affects the signaling of both complexes. As it has been previously shown loss of Tsc1 results in increased mTORC1 activity and decreased mTORC2. We found that reduction of Raptor, but not Rictor, rebalances both mTORC1 and mTORC2 signaling and improves the morphology of Tsc1 knock-out neurons in vitro. We also observed that Raptor reduction in vivo, was sufficient to prevent several neurologic phenotypes in a mouse model of TSC, including mTORC1 hyperactivity, neuronal hypertrophy, demyelination, network hyperactivity and premature mortality. Finally, we examined Raptor manipulation as a therapeutic strategy by postnatally injecting shRNA in mice with embryonic loss of Tsc1. We found that postnatal Raptor downregulation can rescue both cell and non-cell autonomous mechanisms including mTORC1 hyperactivity, neuronal hypertrophy, and myelination. We also found that shRptor can significantly extend survival and improve the overall development of Tsc1-KO mice. Notably, when we examined the effects of Raptor manipulation as therapeutic strategy for TSC-related seizure activity we found that downregulation of Raptor did not improve the phenotype. Interestingly, neither rapamycin treatment was able to rescue this phenotype suggesting that this seizure like activity in our in vitro model, cannot be rescued via mTORC1 suppression after it has been established. Overall, this thesis provides novel insights in the regulation and function of the mTOR pathway in neurons. We identify mTORC1 as the complex that drives neurologic manifestations in mouse models of TSC and propose that Raptor manipulation could be a promising therapeutic strategy for TSC and potentially other mTORopathies. We have also established an in vitro model where we can study TSC related seizure-like activity. This model reveals that cell-autonomous changes that drive neuronal hyperactivity due to Tsc1 loss can become mTORC1 independent over time. Together this data generates new insights that will aid in understanding more in depth the molecular mechanisms that drive TSC neurologic manifestations and provide important information for the development of novel preventative and therapeutic strategies.