mTORC1 and mTORC2 regulate distinct aspects of glutamatergic synaptic transmission

Abstract

25Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in 26 neurons it regulates synaptic transmission, which is thought to be a major mechanism through which 27 altered mTOR signaling leads to neurological disease. Although previous studies have delineated 28 postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, 29 the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR's role in 30 synaptic transmission may be complex-specific. To better understand each complex's role in synaptic 31 transmission, we genetically inactivated mTORC1 or mTORC2 in cultured mouse glutamatergic 32 hippocampal neurons. Inactivation of either complex reduced neuron growth and evoked EPSCs, 33 however, mTORC1 exerted its effects on eEPSCs at the postsynapse and mTORC2 at the presynapse. 34 Furthermore, inactivation of each complex altered specific modes of synaptic vesicle release, suggesting 35 that mTORC1 and mTORC2 differentially modulate postsynaptic responsiveness and presynaptic release 36 to optimize glutamatergic synaptic transmission. 37 38 39 40 41 42 43 44 45 103 Results 104 Inactivation of mTORC1 or mTORC2 in neurons, via Raptor or Rictor deletion, causes distinct effects on 105 downstream mTOR signaling. 106To investigate the effects of mTORC1 or mTORC2 inactivation on neurons, we cultured primary 107 hippocampal neurons isolated from P0-P1 Raptor flox/flox or Rictor flox/flox mice on astrocytes isolated from 108 P0-P1 wild-type mice. At the time of plating, we transduced the neurons with adeno-associated viruses 109 (AAVs) expressing either an mCherry-Cre fusion protein or mCherry alone, both driven by the hSyn 110 promoter, to generate knockout (Raptor-KO or Rictor-KO) or control (Raptor-Con or Rictor-Con) 111 neurons, respectively. To assess the efficiency of gene deletion in Raptor-KO and Rictor-KO neurons, we 112 performed quantitative immunofluorescence analysis for phospho-S6 (S240/244) (pS6), an indicator of 113 mTORC1 activity, and phospho-Akt (S473) (pAkt), an indicator of mTORC2 activity. In Raptor-KO neurons, 114 the mean fluorescence intensity of the pS6 signal was decreased relative to Raptor-Con neurons (Con: 1151.00 ± 0.05, Rap-KO: 0.25 ± 0.03, p < 0.001; Figure 1A1-3), verifying a reduction in mTORC1 activity. The 116 pAkt signal was, however, increased in Raptor-KO neurons relative to Raptor-Con neurons (Con: 1.00 ± 117