Tanc2-dependent direct and regulated mTOR inhibition balances mTORC1/2 signaling in developing mouse and human neurons


 mTOR signaling, involving mTORC1 and mTORC2 complexes, critically regulates neural development and is implicated in various brain disorders. mTORC1/2 components that stimulate mTOR kinase activity strongly affect neurodevelopment, but mTOR-inhibitory mTORC1/2 components do not, questioning the role of balanced mTOR regulation in neurodevelopment. We found 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 directly interacts with and inhibits mTOR, which is suppressed by mTOR-activating serum or ketamine, a fast-acting antidepressant. Tanc2 and Deptor, known to inhibit mTORC1/2 but minimally affect neurodevelopment, distinctly inhibit mTOR in early- and late-stage neurons. Patient-derived Tanc2 mutations disable Tanc2 function, and human Tanc2 inhibits mTORC1/2. Therefore, Tanc2 represents a novel mTORC1/2 inhibitor with strong neurodevelopmental impacts, implicating mTOR inhibition in treating TANC2-related brain disorders and Tanc2 modulation in treating mTOR-related disorders.

Functionally, the homozygous deletion of Tanc2 in mice leads to embryonic lethality 16 . In humans, TANC2 mutations are extensively associated with various neuropsychiatric disorders, including intellectual disability, ASD, developmental delays, and schizophrenia 17,22−30 . Disruptive TANC2 mutations were recently identi ed in 20 different patients with neurodevelopmental symptoms associated with psychiatric disorders 17 . These results suggest that Tanc2 is a critical regulator of brain development and function, but the underlying mechanisms remain unclear.
In behavioral tests, adult male Tanc2 +/mice (2-5 months; male) showed impaired spatial learning and memory in the Morris water maze, but normal novel-object recognition ( Fig. 1b; Supplementary Fig. 1a). These mice also displayed hyperactivity and anxiolytic-like behavior, but largely normal social and depression-like behavior, and as neonates, showed suppressed ultrasonic vocalizations upon mother separation ( Supplementary Fig. 1b-e and 2). Female adult Tanc2 +/mice showed behavioral abnormalities similar to those of males ( Supplementary Fig. 3). These results indicate that Tanc2 +/mice are more relevant to human disease conditions.
The abovementioned decrease in LTD at 3-4 weeks, which contrasts with the normal LTP at a similar age (4-5 weeks), cannot be explained by the decrease in currents of NMDA receptors (NMDARs), which are known to regulate both LTP and LTD 31,32 . We thus tested whether synaptic signaling downstream of NMDAR activation, also known to control LTP/LTD 31,32 , is altered by immunoblot analysis of neuronal signaling proteins. mTOR hyperactivity in Tanc2-mutant mice Intriguingly, mTOR activity, measured by mTOR phosphorylation (S2448) in immunoblot analyses, was markedly (~5-fold) increased in the whole brain of Tanc2 +/pups (P14) without a change in total mTOR levels ( Fig. 1e). This change was accompanied by hyper-phosphorylation of 4E-BP (T37/46), a downstream target of mTOR 1-4 , but not S6 (S235/236), another mTOR target [1][2][3][4] , likely owing to compensatory changes occurring in heterozygous mice (see the stronger changes induced by homozygous Tanc2 deletion, below). In contrast, activities of PI3K (phosphoinositide 3-kinase), PTEN (phosphatase and tensin homolog) and TSC1/2 (tuberous sclerosis 1/2)-signaling proteins upstream of mTOR-were normal ( Supplementary Fig. 5a), suggesting that they do not contribute to the mTOR hyperactivity.
Early rapamycin treatment normalizes LTP and behaviors in adult Tanc2 +/mice To gain mechanistic insight into how Tanc2 deletion induces mTOR hyperactivity, we rst tested whether Tanc2 directly interacts with mTOR using protein-protein binding assays. Puri ed Tanc2 protein directly interacted with puri ed mTOR protein (Fig. 3a). Tanc2 also formed a complex with mTOR in the mouse brain (Fig. 3b,c). This interaction was mediated by multiple regions of Tanc2 protein and the C-terminal region of mTOR containing FRB and kinase domains ( Fig. 3d-f). Here, mTOR was found to additionally interact with Tanc1, a relative of Tanc2 that is strongly expressed in late stages of rat brain development (>P14) and regulates synapse development, but is not critical for mouse development 15,16 .
The results described thus far suggest that Tanc2 directly interacts with mTOR, but do not speak to whether Tanc2 inhibits the kinase activity of mTOR. We tested this possibility by overexpressing Tanc2 in HEK293T cells, and found that this was su cient to inhibit endogenous mTOR activity ( Supplementary  Fig. 7). Consistent with this, in vitro assays using puri ed proteins showed that Tanc2 directly inhibits mTOR kinase activity, as evidenced by decreased phosphorylation of the mTORC1 (mTOR + Raptor) target S6K in the presence of Tanc2 (Fig. 3g).

Serum and ketamine regulate the Tanc2-mTOR interaction
We next investigated whether Tanc2-mTOR interactions are regulated by extracellular in uences, rst testing serum, which is known to activate mTOR 2 . Serum starvation promoted the colocalization and biochemical association of Tanc2 with mTOR in HEK293T cells within ~4 hours. This effect was reversed by serum replenishment for ~24 hours (Fig. 4a,b), suggesting that mTOR dissociates from Tanc2 upon serum stimulation. Moreover, the Tanc2-mTOR interaction induced by serum starvation was inhibited by rapamycin ( Fig. 4c,d), suggesting that Tanc2 and rapamycin compete for binding to the mTOR FRB domain. Tanc1, which also associates with mTOR in the brain, interacted with mTOR in a serum-and rapamycin-dependent manner ( Supplementary Fig. 8).
Tanc2, Deptor, and Tanc1 distinctly inhibit mTORC1/2 in early-and late-stage neurons Because Deptor, similar to Tanc2, also binds and inhibits mTORC1/2 35 , we tested whether Tanc2 and Deptor show overlapping or distinct spatiotemporal expression patterns. Immunoblot analyses using cultured neurons and mouse brain extracts showed that Tanc2 protein was more strongly expressed in early stages (embryonic and early postnatal) and was less enriched at synapses (Supplementary Fig. 10). In contrast, Deptor and Tanc1 showed progressive increases in expression across postnatal stages and stronger synaptic enrichment in both cultured neurons and mouse brains, a pattern similar to that reported for rat Tanc1 and Tanc2 15,16 .
To determine whether neurons or glial cells are more important for Tanc2-dependent mTOR inhibition, we selectively knocked down Tanc2 in neuron-or glia-enriched early-stage cultured hippocampal neurons (DIV7-14). Neuronal, but not glial, Tanc2 knockdown induced mTOR hyperactivity, and, in line with this, Tanc2 expression was much weaker in glial cells ( Supplementary Fig. 11), suggesting that Tanc2 is more important for mTOR inhibition in neurons than in glial cells at early stages.

Patient-derivedTanc2 mutations suppress Tanc2-dependent mTOR inhibition
To determine whether there is a relationship between Tanc2-dependent mTOR inhibition and human brain disorders, we rst tested whether speci c Tanc2 mutations associated with intellectual disability, schizophrenia, and ASD identi ed in humans (R760C, A794V, and H1689R) 22,23,26 affected interactions with mTOR or inhibition of mTOR activity (Fig. 6a). Coimmunoprecipitation experiments showed that, of these mutants, only Tanc2-H1689R failed to biochemically associate with mTOR in HEK293T cells (Fig.   6b,c); however, all three Tanc2 mutants failed to inhibit mTOR activity (Fig. 6d-f). Therefore, patientderived Tanc2 mutations disrupt the mTOR-binding and/or mTOR-inhibitory activity of Tanc2.

TANC2 in human neurons inhibits mTORC1 and mTORC2
Finally, we tested whether Tanc2 inhibits mTOR activity in human neurons. To this end, we knocked down TANC2 in human neural progenitor cells (NPCs) developing into mature neurons for 2 weeks using two independent TANC2 knockdown constructs. Both TANC2 knockdown constructs similarly increased phosphorylation of S6 (S235/236), 4E-BP (T37/46), and GSK3b (S9), although they exerted mixed effects on Akt (S473) phosphorylation (Fig. 6g-i). mTOR phosphorylation was unaltered, similar to the results from mouse neurons (Fig. 5). These results collectively suggest that Tanc2 inhibits mTORC1/2 in both human and mouse neurons.

Discussion
The present study suggests that Tanc2 is a novel and regulated mTOR inhibitor that has strong neurodevelopmental impacts and therapeutic potential. The rst important conclusion from our results is that Tanc2 binds to mTOR. In support of this, Tanc2 forms a complex with mTOR in heterologous cells and in the mouse brain. More directly, puri ed Tanc2 proteins form a complex with puri ed mTOR proteins. Tanc2 uses its multiple domains to associate with mTOR, whereas mTOR binds to Tanc2 through its C-terminal region, containing the FRB, kinase, and FATC domains. The latter is further supported by that rapamycin, known to bind to the FRB domain of mTOR, blocks the colocalization and biochemical association between Tanc2 and mTOR. This result suggests the possibility that rapamycin could be used to block the Tanc2-mTOR interaction and to promote mTOR activity in various contexts such as decreased mTOR activity in human disorders.
Tanc2 binds to mTOR in a regulated manner. The presence of serum, well known to activate mTOR, weakens the colocalization and biochemical association between Tanc2 and mTOR. In addition, ketamine, a fast-acting antidepressant known to promote excitatory synapse functions and mTOR activity 34 , inhibits the Tanc2-mTOR interaction in the mouse brain. These results suggest that Tanc2 inhibits mTOR in a regulated manner to coordinate mTOR activity under various nutritional states and during brain development and neuronal or synaptic activities. Speci c mechanisms that underlie the regulated Tanc2-mTOR interactions remain to be determined although they could be posttranslational modi cations of mTOR or Tanc2 at binding interfaces or regulatory domains.
Perhaps the most important conclusion of the present study is that Tanc2 inhibits mTOR. This is supported by multiple lines of in vitro and vivo evidence. Most directly, puri ed Tanc2 inhibits the kinase activity of mTOR, as shown by the suppression of mTOR-dependent phosphorylation of an mTOR substrate (S6K). In addition, Tanc2 overexpressed in HEK293T cells inhibits mTOR, while mutant Tanc2 proteins carrying human mutations fail to bind and inhibit mTOR. Moreover, acute knockdown of Tanc2 increases mTOR activity in cultured mouse neurons at around developmental stages of strong Tanc2 expression. Tanc2 +/− mice show increased mTOR activity in both mTORC1 and mTORC2 complexes. In addition, cre-dependent acute knockout of Tanc2 in an independent Tanc2-mutant mouse line increases mTOR activity in mTORC1/2. In human neurons, Tanc2 knockdown increases mTOR activity in mTORC1/2 in neural progenitor cells developing into neurons. These results strongly suggest that Tanc2 binds to and inhibits mTOR in mouse and human neurons at early stages.
In addition to Tanc2, Tanc1 interacts with and inhibits mTOR in a rapamycin-dependent manner. Tanc1 expression sharply increases during postnatal stages of mouse brain development, whereas Tanc2 expression is relatively stronger at earlier stages. Deptor, a known mTOR inhibitor, also shows strong latestage expression, similar to Tanc1. It is therefore possible that Tanc2, Tanc1, and Deptor distinctly inhibit mTOR across different developmental stages. Indeed, our results indicate that Tanc2 and Tanc1/Deptor inhibit mTOR more strongly at around postnatal weeks 2 and 4, respectively. These results are in line with the differential impacts of homozygous Tanc2 and Tanc1/Deptor deletions in mice, where the deletion of Tanc2, but not Tanc1 or Deptor, leads to embryonic lethality 14,16 .
Tanc2 and Tanc1 interact with the PSD-95 family of scaffolding proteins, known to mediate the molecular organization of multi-protein complexes at cell-to-cell junctions such as neuronal synapses in order to couple receptor activations with signaling pathways 18,19 . Therefore, Tanc2 and Tanc1 may recruit mTORC1/2 complexes to PSD-95-based multiple protein complexes at excitatory postsynaptic sites. In line with this idea, Tanc2 has been suggested to recruit cargo dense core vesicles driven by the KIF1A motor protein to excitatory synapses 21 . Synaptically localized mTORC1/2 may be inhibited by local Tanc2 until mTOR activity is increased by the activation of synaptic receptors such as TrkB and mGluRs 36 . The four known members of the PSD-95 family (PSD-95, PSD-93, SAP102, and SAP97) display differential spatiotemporal expression patterns; i.e. PSD-95 and PSD-93 are more abundant at later developmental stages whereas SAP102 expression is stronger at earlier stages. It is therefore possible that Tanc2 and Tanc1 may coordinate mTORC1/2 signaling at both synaptic and non-synpatic sites of PSD-95-enriched multi-protein complexes in developing neural and non-neural tissues.
The synaptic and behavioral phenotypes of Tanc2 +/− mice implicate Tanc2 in the regulation of synaptic plasticity and behaviors, including LTP, learning and memory, hyperactivity, and anxiety-like behavior, all of which are reversed by rapamycin-dependent mTOR inhibition. In humans, Tanc2 mutations have been extensively associated with various neurodevelopmental and neuropsychiatric disorders, including intellectual disability, schizophrenia, and ASD 17,22−30 . These results, together with the embryonic lethality in Tanc2 −/− mice and strongly increased mTOR activity in Tanc2 +/− mice, suggest that Tanc2 regulates normal bran development and function by coordinating mTOR inhibition, and that rapamycin-dependent mTOR inhibition could be used to treat human patients with Tanc2 mutations and resulting mTOR hyperactivity. In addition, modulation of Tanc2 activity, i.e. anti-sense Tanc2 knockdown or virusmediated Tanc2 overexpression, could be used to treat various mTOR-related brain disorders 5-7,37−39 . These therapeutic potentials extend to non-neural tissues and non-brain mTOR-related disorders such as metabolic diseases 1,2 because Tanc2 and Tanc1 are expressed in various non-neural tissues in mice and humans (www.ebi.ac.uk/gxa/home) 16,17 .
In conclusion, our study reports that Tanc2 is a novel and regulated mTOR inhibitor with strong neurodevelopmental impacts, supporting the general notion that balanced mTOR regulation involving both mTOR activators and inhibitors is important for normal brain development and function. In addition, our results suggest that mTOR inhibition could be an effective strategy for treating human individuals with TANC2 mutations suffering from neuropsychiatric disorders, including intellectual disability, ASD, developmental delays, and schizophrenia. In addition, Tanc2 modulations promoting or suppressing mTOR signaling could have therapeutic potential for the treatment of various mTOR-related peripheral and brain disorders.  Early, chronic rapamycin treatment improves impaired spatial learning and memory in Tanc2+/-mice Tanc2 directly interacts with and inhibits mTOR (a) Puri ed Tanc2 protein directly interacts with puri ed mTOR protein. GST-Tanc2 (full-length) protein was used to pull down puri ed mTOR protein. Input, 20%. (b and c) Tanc1 and Tanc2 form a complex with mTOR in the mouse brain. Whole-brain lysates (P14; mouse) were immunoprecipitated (IP) with pan-Tanc or mTOR antibodies, followed by immunoblotting.
Note that mTOR pull-down also coprecipitated PSD-95 through Tanc2. (d) Both N-terminal and C-terminal regions of puri ed Tanc2 protein directly interact with puri ed mTOR. GST-tagged puri ed N-and Cterminal regions of human Tanc2 (aa 1-1358 and aa 1359-1990) were coupled to glutathione beads and incubated with puri ed mTOR proteins, followed by GST pull-down and immunoblot analysis. (e) Tanc2 forms a complex with mTOR in HEK293T cells through multiple regions of Tanc2. Lysates of HEK293T cells expressing deletion variants of Flag-Tanc2 (near-full-length, aa 127-1990; N-terminal region, aa 127-835; middle region, aa 836-1358; C-terminal region, aa 1234-1990) and mTOR (endogenous) were immunoprecipitated with Flag antibodies and immunoblotted with anti-Flag (for Tanc2) and mTOR antibodies. Note that all four deletion variants of Tanc2 interacted with mTOR, suggesting that multiple regions of Tanc2 are involved in mTOR binding. We used the near full-length Tanc2 because the fulllength construct was unavailable at the time of the experiment; experiments repeated using the full-length  Serum and ketamine regulate the interaction of Tanc2 with mTOR. (a) Serum starvation induces colocalization of Tanc2 and mTOR in HEK293T cells, an effect that is reversed by serum re-feeding. HEK293T cells transfected with CFP-Tanc2 + YFP-mTOR were subjected to serum starvation (-serum) to inactivate mTOR, or to no serum starvation (control; +serum), for 4 hours followed by serum re-feeding for 24 hours while checking changes at 4-hr and 24-hr time points. The colocalization was quanti ed using Pearson's correlation analysis of colocalized pixels (see Methods for details). (b) Rapamycin blocks serum starvation-induced Tanc2-mTOR colocalization in HEK293T cells. HEK293T cells expressing CFP-Tanc2 and YFP-mTOR were treated with rapamycin or vehicle for 2 hours before starting serum starvation. Colocalization was quanti ed by Pearson's correlation analysis of colocalized pixels (see Methods for details). (n = 24 cells from 3 independent experiments. ***P < 0.001, ns, not signi cant, Student's t-test). Scale bar, 10 µm. (c) Increased biochemical association between Tanc2 and mTOR induced by serum starvation in HEK293T cells, as determined by coimmunoprecipitation (coIP). HEK293T cells expressing Flag-Tanc2 and mTOR (endogenous) in the presence and absence of serum starvation (4 hours) were immunoprecipitated with Flag antibody (for Tanc2) and immunoblotted as indicated. The lower Flag-Tanc2 band represents a degradation product. mTOR signals were normalized to Tanc2 signals for quanti cation. (n = 4 independent experiments, *P < 0.05, Student's test). (d) Rapamycin blocks the serum starvation-induced biochemical association of Tanc2 with mTOR in HEK293T cells, as determined by coimmunoprecipitation. HEK293T cells expressing Flag-Tanc2 and mTOR (endogenous) were treated with rapamycin or vehicle for 2 hours before starting serum starvation, followed by immunoprecipitation with anti-Flag antibodies (for Tanc2) and immunoblotting, as indicated. mTOR signals were normalized to Tanc2 (Flag) signals for quanti cation. (n =3 independent experiments, ***P < 0.01, ns, not signi cant, Student's test). (e) Reduced biochemical association between Tanc1/2 and mTOR in the mouse brain (P13-14) upon ketamine treatment (10 mg/kg; i.p.), as shown by coIP experiments on whole-brain crude synaptosomes from ketamine-treated and -untreated mice using pan-Tanc or mTOR antibodies, followed by immunoblotting. Raptor was immunoblotted to show mTOR activation, and PSD-95 was immunoblotted for coIP with Tanc1/2 (positive control). (n = 4 independent experiments, *P < 0.05, ns, not signi cant, one-way ANOVA with Bonferroni test).