Tanc2-mediated mTOR inhibition balances mTORC1/2 signaling in the developing mouse brain and human neurons

mTOR signaling, involving mTORC1 and mTORC2 complexes, critically regulates neural development and is implicated in various brain disorders. However, we do not fully understand all of the upstream signaling components that can regulate mTOR signaling, especially in neurons. Here, we show a direct, regulated inhibition of mTOR by Tanc2, an adaptor/scaffolding protein with strong neurodevelopmental and psychiatric implications. While Tanc2-null mice show embryonic lethality, Tanc2-haploinsufficient mice survive but display mTORC1/2 hyperactivity accompanying synaptic and behavioral deficits reversed by mTOR-inhibiting rapamycin. Tanc2 interacts with and inhibits mTOR, which is suppressed by mTOR-activating serum or ketamine, a fast-acting antidepressant. Tanc2 and Deptor, also known to inhibit mTORC1/2 minimally affecting neurodevelopment, distinctly inhibit mTOR in early- and late-stage neurons. Lastly, Tanc2 inhibits mTORC1/2 in human neural progenitor cells and neurons. In summary, our findings show that Tanc2 is a mTORC1/2 inhibitor affecting neurodevelopment.


Supplementary figures and legends
Supplementary Fig. 1. Hyperactivity and moderate anxiolytic-like behavior in male Tanc2 +/mice.
(a) Normal novel object-recognition memory in Tanc2 +/mice (2-5 months; male) in the novel object-recognition test, as shown by novel object preference (% time spent in exploring a novel object relative to the total time spent exploring both familiar and novel objects). F, familiar object; N, new object. Note that these mice also showed hyperactivity in the novel object arena, as supported by the distance moved and movement velocity. The increased frequency of novel object exploration in the mutant mice might be attributable to the increased hyperactivity. Data: minimal,maximal,median,25%,and 75% values. (n = 17 [WT,HT], *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, Student's t-test).
(b) Hyperactivity of Tanc2 +/mice (2-5 months; male) in the open-field test, as shown by distance moved over 60 minutes and total distance moved. Note also that the time spent in the center region of the open field arena was normal in Tanc2 +/mice, indicative of normal anxiety-like behavior. Data: mean ± SEM (line graphs), minimal,maximal,median,25%,and 75% values. (n = 13 [WT], 11 [HT], **P < 0.01, ns, not significant, two-way RM ANOVA and Student's t-test).
(d) Moderate anxiolytic-like behavior of Tanc2 +/mice (2-5 months; male) in the elevated plus-maze test, as shown by time spent in open and closed arms. There was no genotype difference in the total distance moved. Data: minimal,maximal,median,25%,and 75% values. (n = 13 [WT], 11 [HT], *P < 0.05, ns, not significant, two-way RM ANOVA with Bonferroni test and Student's t-test).
See Source Data 1 for raw data values and Supplementary Table 1 for statistical details.
See Source Data 1 for raw data values and Supplementary Table 1 for statistical details. Supplementary Fig. 3. Female Tanc2 +/mice display hyperactivity and anxiolytic-like behavior, but normal depression-like behavior.
(a) Hyperactivity of female Tanc2 +/mice (2-5 months) in the open-field test, as shown by distance moved over 60 minutes and total distance moved. Note also that the time spent in the center region of the open field arena was normal in female Tanc2 +/mice, indicative of normal anxiety-like behavior. Data: mean ± SEM (line gr aphs), minimal, maximal, median, 25%, and 75% values. (n = 16 mice [WT], and 11 mice [HT], **P < 0.01, ns, not significant, two-way RM ANOVA and Student's t-test).
(c) Normal anxiety-like behavior of female Tanc2   (a) Serum starvation (4 hours) promotes the interaction between Tanc1 and mTOR in heterologous cells, as demonstrated by coimmunoprecipitation. HEK293T cells expressing Flag-Tanc1 (human) were incubated with serum (or no serum; control) for 4 hours prior to coimmunoprecipitation and immunoblot experiments. mTOR signals were normalized to Tanc1 signals for quantification. Data: mean ± SD. (n = 4 independent experiments, *P < 0.05, Student's t-test).
(b) Rapamycin inhibits serum starvation-induced increases in the interaction between Tanc1 and mTOR in heterologous cells. Flag-Tanc1 was expressed in HEK293T cells in the presence of serum. Pretreatment of cells with rapamycin for 2 hours was followed by serum starvation for 4 hours; cell lysates were subjected to coIP and immunoblot experiments. mTOR signals were normalized to Tanc1 signals for quantification. Data: mean ± SD. (n = 3 independent experiments, ***P < 0.05, ns, not significant, Student's t-test).
See Source Data 1 for raw data values and Supplementary Table 1 for statistical details. Supplementary Fig. 6. Ketamine induces changes in mTOR activity and synaptic levels of Tanc2-associated and mTORC proteins in the mouse brain.
(a and b) Ketamine induces mTOR activation in the mouse brain (P13-14; 10 mg/kg; i.p.), as shown by the time-dependent increases in the phosphorylation of mTOR and the mTORC1 protein Raptor in crude synaptosomes. Note that synaptic levels of other mTORC1/2 proteins (PRAS40 and Deptor) and PSD-95 also increased, whereas those of Tanc2 (but not Tanc1) were modestly decreased. Data: mean ± S D. (n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, one-way ANOVA with Bonferroni test).  Fig. 7. Temporal changes in total levels and synaptic enrichment of Tanc2, Deptor, and Tanc1 in cultured mouse hippocampal neurons and the mouse brain.
(a) Western blot analyses of whole lysates from cultured mouse hippocampal neurons show Tanc2 protein levels that are largely stable across developmental stages (DIV/days in vitro 7-28) and stronger relative to Tanc1 at early stages, while Deptor and Tanc1 protein levels gradually increase. Western blot analyses of crude synaptosomes from cultured mouse hippocampal neurons show largely unchanged synaptic levels of Tanc2 proteins across the developmental stages, which contrasts with the increasing synaptic levels of Deptor and Tanc1 proteins. Total and synaptic levels of PRAS40 (a component of mTORC1) were largely unchanged across developmental stages. PSD-95 was used as a positive control for stage-dependent increases in total expression and synaptic enrichment, and β-actin was used as a loading control.
(b and c) Western blot analyses of whole lysates from the mouse brain (in vivo results) show Tanc2 protein levels that are largely stable across developmental stages (E/embryonic day 18-P/postnatal day 28) and higher relative to Tanc1 at early stages, while Deptor and Tanc1 protein levels gradually increase. Western blot analyses of crude synaptosomes from the mouse brain show decreasing synaptic levels of Tanc2 proteins across the developmental stages, which contrasts with the increasing synaptic levels of Deptor and Tanc1 proteins. Total and synaptic levels of PRAS40 were largely unchanged across developmental stages. PSD-95 was used as a positive control for stage-dependent increases in total expression and synaptic enrichment, and β-actin was used as a loading control. Data: mean ± SD. (n = 4 independent experiments).  Fig. 8. Acute Tanc2 knockdown in cultured mouse neurons, but not glial cells, leads to mTORC1/2 hyperactivity.
(c) Control co-immunofluorescence staining showing that cultured neurons are positive for NeuN (neuronal marker) but not for GFAP (astrocytic marker) and that cultured glia are positive for GFAP but not for NeuN. DAPI was used for nuclear staining. Scale bar, 50 µm. Data: mean ± SEM. (n = 13 images for neuron and glial cultures, ***P < 0.001, Student's t-test).   (a) Morphological characterization of pan-NPCs by immunofluorescence staining for Nestin and SOX2 (NPC markers) and pan-neurons at DIV7 and DIV14 by staining for MAP2 and Tuj1 (neuronal markers). Scale bar, 20 µm. Three independent experiments yielded similar results.