Disentangling mTOR complexes underlying PTEN-loss-associated Autism Spectrum Disorder

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a negative regulator of the AKT serine threonine kinases (Protein Kinase B) in the mechanistic target of rapamycin (mTOR) signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten-loss results in neuronal hypertrophy, hyperexcitability, seizures, and social deficits. The underlying molecular mechanisms of these phenotypes are not well-delineated. I aimed to disentangle the role of the downstream mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) in orchestrating aberrations in neuronal form and function resulting from Pten-loss. First, we found that the rapamycin-mediated inhibition of mTORC1 in dentate gyrus granule neurons prevented the increase in soma size, migration, spine density and dendritic overgrowth due to lack of Pten. Regulatory-associated protein of mTOR (Raptor) is a scaffolding protein for the formation of themTORC1 complex. Postnatal genetic knockout (KO) of Raptor to disrupt mTORC1 complex formation blocked neuronal hypertrophy observed with Pten-loss. Electrophysiological recordings from cultured neurons revealed that genetic disruption of mTORC1 rescued the increase in excitatory synaptic transmission observed with loss of Pten. Thus, we have identified an essential role for mTORC1 in orchestrating the observed neuronal hypertrophy and synapse formation. Second, as rapamycin-insensitive companion of mTOR (Rictor) is a scaffolding protein for formation of the mTORC2 complex, I found that knocking-out Rictor gene is sufficient to restore normal levels of spine density, and distal dendritic growth. Rictor KO partially rescued somatic hypertrophy, migration deficits, and proximal dendritic branching resulting from Pten-loss. Simultaneous disruption of both mTORC1 and mTORC2 did not result in an additive rescue of somatic hypertrophy, suggesting a role for mTORC1 in the rescue mediated by mTORC2. mTORC2-driven phosphorylation of AKT proteins, a family of kinases upstream of mTORC1, provides one such point of convergence. Disruption of mTORC2 decreased phosphorylation of wildtype AKT3 but did not rescue the morphological effects seen on reconstitution with a phosphomimetic AKT3. Taken together, the data suggest that the rescue of the somatic hypertrophy of Pten KO neurons resulting from Rictor-loss is dependent on the phosphorylation of AKT3. Loss of this phosphorylation downregulates the activity of mTORC1, resulting in a partial restoration of somatic hypertrophy of neurons lacking Pten. Lastly, I found that pharmacologically decreasing the rate of microtubule (MT) polymerization in granule neurons is sufficient to reduce dendritic overgrowth in vivo, as well as to improve the spatial navigation and memory deficits of Pten mutant mice. By teasing apart the molecular signaling underlying neuronal alterations associated with Pten-loss, I have provided mechanistic insight and identified potential new targets for development of therapeutics for autism spectrum disorder