Evaluation of hyperactive mTORC1 signaling in the hippocampus of Tsc1 heterozygous mice: Implications for the fragile X mental retardation protein

Altered mammalian target of rapamycin complex 1 (mTORC1) signaling is a feature of a number of neurodevelopmental disorders that display high rates of mental retardation with comorbid autistic features. For example, mTORC1 signaling is decreased in animal models of Rett syndrome and increased in phosphatase and tensin homolog (PTEN) hamartoma tumor syndromes, neurofibromatosis, fragile X syndrome and tuberous sclerosis complex (TSC). TSC is the canonical "mTORopathy" and is due to mutations in either TSC1 or TSC2, which are upstream regulators of mTOR kinase activity in mTORC1. Most patients are born heterozygous for either TSC gene and sustain additional inactivating mutations during development leading to loss of heterozygosity. TSC1 and TSC2 are ubiquitously expressed in a complex with Tre2-Bub2-Cc6 1 domain family member 7 (TBC1 D7), however only mutations of TSC1 and TSC2 have been shown to cause TSC. The TSC1/TSC2 complex acts as a molecular brake towards mTORC1 signaling and loss of heterozygosity leads to increased mTORC1 activity that drives the formation of cortical malformations and slow growing tumors.;These malformations, and associated seizure activity, clearly contribute to impaired cognition however imaging and neurocognitive studies suggest that they are not sufficient to fully explain the cognitive impairment observed in TSC patients. This is further suggested by animal models in which Tsc1 or Tsc2 heterozygosity is sufficient to impair neuroplasticity and learning and memory despite the absence of brain malformations, tumors, and clinical seizures. Physiological and behavioral impairments in Tsc2+/- mice are rescued by rapamycin treatment (an inhibitor of mTORC1) indicating that they are reversible and mTORC1-dependent. These findings point to a biochemical and molecular basis for cognitive deficits in TSC. One candidate molecule that is involved in neuroplasticity and could contribute to physiological and behavioral symptoms in TSC animal models is the Fragile X Mental Retardation Protein (FMRP), which is reportedly downstream of mTORC1.;FMRP is an mRNA-binding protein that regulates the translation of ∼4-6% of brain mRNAs, many of which modulate neuroplasticity. Mutation of FMR1 (the X-linked gene encoding FMRP) results in fragile X syndrome (FXS), the leading cause of inherited intellectual disabilities and autism. FMRP regulation of neuroplasticity is thought to be dictated by FMRP phosphorylation at serine 499 (S499) resulting in FMRP association with stalled (inactive) polyribosomes and translational repression of mRNA. The kinase responsible for FMRP 5499 phosphorylation in mice (S500 in humans) was identified as the mTORC1-dependent ribosomal protein S6 kinase 1 (S6K1). Considering that S6K1 sits directly downstream of mTORC1 and is likely hyperactive in Tsc2 +/- hippocampi and Tsc1+/- Purkinje cells (Tsai et al., 2012), S6K1 may link elevated mTORC1 activity to altered FMRP phosphorylation and function in the Tsc1 heterozygous state.;We thus set out to investigate S6K1 activity, as well as FMRP S499 phosphorylation, in Tsc1 mouse models of TSC. Surprisingly, we found that FMRP S499 phosphorylation is unchanged in heterozygous and conditional Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Subsequent experiments revealed that neither up- nor down-regulation of the mTORC1-S6K1 axis, either in vivo or in vitro, has any effect on FMRP S499 levels. Furthermore, S6K1 is dispensable for FMRP S499 phosphorylation in vivo, indicating that the mTORC1-S6K1 pathway plays no role in regulating FMRP S499 phosphorylation.