Impaired mTOR Macroautophagy and Neurocognitive Deficits in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder caused by mutations in either the Tsc1 or Tsc2 gene. More than 50% of TSC patients exhibit mental retardation and cognitive delay. The remaining patients, though with IQ in the normal range, often present difficulties with long-term memory and executive skills. Current treatments to combat neurocognitive symptoms in TSC are largely ineffective. Recent evidence suggests that disinhibited mTOR signaling contributes to TSC-related learning and memory deficits, and that the mTOR inhibitor rapamycin corrects both synaptic and cognitive defects in Tsc1 and Tsc2 heterozygous mutant mice. Clinical trials are thus underway to test the effects of rapamycin analogues, e.g., everolimus, on neurocognitive problems in TSC patients. One caveat for rapamycin treatment is that long-term therapy is required to maintain effectiveness, which may cause adverse effects, e.g., infections, hyperlipidemia, thrombocytopenia, and disrupted brain development and synaptic plasticity. The efficacy of rapamycin can moreover be compromised by a negative feedback loop from mTOR/S6K1 to the upstream mTOR regulators PI3K/Akt kinases. In this study, we will identify mTOR-downstream molecules or pathways responsible for TSC-related cognitive deficits, with the goal of developing more specific treatment targeting downstream elements of the mTOR pathway, thus limiting side effects of mTOR inhibitors. Macroautophagy (autophagy hereafter) is a lysosomal degradation process downstream of mTOR that maintains protein quality control via the degradation of cellular proteins and organelles to generate amino acids. We recently discovered that autophagy is downregulated by hyperactive mTOR in Tsc1 and Tsc2 deficient mouse brain. Impaired mTOR-autophagy produces synaptic pathology, manifested by increased dendritic spine density and impaired synaptic pruning, a process critical for postnatal synapse maturation. Based on these findings, we hypothesize that autophagy deficiency may underlie synaptic and cognitive dysfunction in TSC by disrupting developmental synapse maturation. We will test this hypothesis in both Tsc2+/- heterozygous mutant and neuroglia Tsc1 knockout (Tsc1mGFAPCreCKO) mouse models. To isolate the effect of autophagy from the other events downstream of hyperactive mTOR, we will specifically deplete the autophagy gene Atg7 in both Tsc2+/- and Tsc1mGFAPCreCKO mice. Cognitive functions will be evaluated in these mice treated with or without rapamycin. Synapse maturation will be assessed by measuring both pre- and post-synaptic functions during period of postnatal synapse development. We will screen for excitatory neuronal and synaptic molecular substrates that are specifically regulated by autophagy during the postnatal synapse development, using innovative technologies including ribosome profiling, recombinant synaptic tagging, and advanced synapse proteomics. The success of this project will further our understanding of the mechanisms that underlie neurocognitive dysfunction in TSC and will promise more targeted downstream therapeutic strategies to reduce or prevent the potentially severe side effects of rapamycin. The identification of molecular changes specific to impaired autophagy may provide drug targets for the treatment of neurocognitive symptoms in TSC bypassing mTOR.