© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs243972. doi:10.1242/jcs.243972

RESEARCH ARTICLE Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis So-Hee Lim1,2, Sangyep Shin3, Myoung-Hwan Kim4, Eung Chang Kim3, Da Yong Lee1, Jeonghee Moon5, Hye-Yeon Park6, Young-Kyoung Ryu6, Young-Mi Kang6, Yu Jeong Kang6, Tae Hwan Kim1, Na-Yoon Lee1, Nam-Soon Kim1, Dae-Yeul Yu7, Insop Shim8, Yoichi Gondo9, Masanobu Satake10, Eunhee Kim2, Kyoung-Shim Kim6,‡, Sun Seek Min3,‡ and Jae-Ran Lee1,‡

ABSTRACT INTRODUCTION PTPRT has been known to regulate synaptic formation and dendritic Depressive disorder is a major health problem with a high prevalence arborization of hippocampal neurons. PTPRT−/− null and PTPRT- and a heavy socioeconomic burden (Levy et al., 2018; Berton and D401A mutant mice displayed enhanced depression-like behaviors Nestler, 2006; Kessler and Bromet, 2013). Although many types of compared with wild-type mice. Transient knockdown of PTPRT in the antidepressants have been developed so far, current antidepressants dentate gyrus enhanced the depression-like behaviors of wild-type still show a delayed onset of action as well as a lack of efficacy (Berton mice, whereas rescued expression of PTPRT ameliorated the and Nestler, 2006; Rush, 2007). At present, many antidepressant drugs behaviors of PTPRT-null mice. Chronic stress exposure reduced are designed to target monoaminergic neurotransmission, but there is a expression of PTPRT in the hippocampus of mice. In PTPRT- growing consensus that altered monoaminergic transmission is not deficient mice the expression of GluR2 (also known as GRIA2) sufficient to explain the etiology of depressive disorder (Berton and was attenuated as a consequence of dysregulated tyrosine Nestler, 2006; Chaudhury et al., 2015; Krishnan and Nestler, 2011; phosphorylation, and the long-term potentiation at perforant– Luscher et al., 2011). Functional imaging studies on patients with dentate gyrus synapses was augmented. The inhibitory synaptic depression show dysregulated neuronal connectivity, and rodent transmission of the dentate gyrus and hippocampal GABA models of depression display neuronal atrophy, reduced synaptic concentration were reduced in PTPRT-deficient mice. In addition, density and cell loss (Zeng et al., 2012; Holmes et al., 2019; Duman the hippocampal expression of GABA transporter GAT3 (also known and Aghajanian, 2012; Czéh et al., 2018). It has also been found that as SLC6A11) was decreased, and its tyrosine phosphorylation was typical antidepressants increase neurotrophic factor expression and increased in PTPRT-deficient mice. PTPRT-deficient mice displayed enhance synaptic plasticity. On the other hand, atypical antidepressants reduced numbers and neurite length of newborn granule cells in the such as ketamine, an NMDA receptor antagonist, increase synaptic dentate gyrus and had attenuated neurogenic ability of embryonic formation and produce rapid antidepressant responses in patients with hippocampal neural stem cells. In conclusion, our findings show that treatment-resistant depression (Machado-Vieira et al., 2012; Jaso et al., the physiological roles of PTPRT in hippocampal neurogenesis, as 2017). The understanding of neuronal loss and synaptic dysfunction well as synaptic functions, are involved in the pathogenesis of mechanisms in depressive disorder could thus lead to the identification depressive disorder. of key factors for the development of fast-acting and effective antidepressants. KEY WORDS: PTPRT, Depression, Knockout, Tyrosine Stressful life events influence the onset and course of depression, but dephosphorylation, Synaptic function, Hippocampal neurogenesis not all people who live a stressful life develop depressive disorder (Caspi et al., 2003). Both genetic and environmental determinants, as well as their interaction, are crucial factors causing depressive disorder (Dunn et al., 2015; Mandelli and Serretti, 2013). The heritability of major depressive disorder has been estimated at ∼30–40%, and 1Rare Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea. 2Department of Biological Sciences, depression is a polygenic trait influenced by many genetic variants Chungnam National University, Daejeon 34134, Korea. 3Department of Physiology (Howard et al., 2018). Recently, genome-wide association studies and Biophysics, School of Medicine, Eulji University, Daejeon 34824, Korea. (GWAS) with large sample sizes (over 100,000 participants) combined 4Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea. 5Disease Target Structure Research Center, Korea Research with publically available genomic data have identified many gene Institute of Bioscience and Biotechnology, Daejeon 34141, Korea. 6Laboratory variants and genetic loci significantly associated with depressive Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea. 7Genome Editing Research Center, Korea disorder (Howard et al., 2019, 2018; Wray et al., 2018; Hyde et al., Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea. 2016; Major Depressive Disorder Working Group of the Psychiatric 8Department of Physiology, College of Medicine, Kyung Hee University, Seoul GWAS Consortium, 2013). These mega-analyses reveal new druggable 02447, Korea. 9Department of Molecular Life Sciences, Tokai University School of Medicine, Shimo-Kasuya, Isehara 259-1193, Japan. 10Department of Molecular genes associated with depression that are not currently associated with Immunology, Institute of Development, Aging and Cancer, Tohoku University, antidepressant treatment. A meta-analysis has identified new genes and Sendai 980-8575, Japan. gene pathways associated with synaptic structure and neurotransmission ‡Authors for correspondence ([email protected]; [email protected]; as well as gene sets associated with depression (Howard et al., 2019). [email protected]) PTPRT (protein tyrosine phosphatase receptor T; also known as RPTPρ) is mainly expressed in the central nervous system and has Y.-M.K., 0000-0002-1988-119X; J.-R.L., 0000-0002-4634-8489 been known to regulate the synaptic formation and dendritic Handling Editor: Giampietro Schiavo arborization of hippocampal neurons (Besco et al., 2006; Lim et al.,

Received 19 January 2020; Accepted 7 September 2020 2009; Park et al., 2012; Lee, 2015). PTPRT has the characteristic Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243972. doi:10.1242/jcs.243972 extracellular ectodomain along with two intracellular catalytic hippocampus of the mice. Inactive PTPRT increased in the domains with tyrosine phosphatase activity. PTPRT forms hippocampus of those mice that were exposed to stress, compared homophilic trans dimers through its extracellular ectodomain, and with levels in control mice (Fig. 2D). These results show that its expression both in pre- and post-synapses can strengthen chronic restraint stress that induces depression-like behaviors synaptic formation. PTPRT appears to obtain easy accessibility to attenuated the expression of PTPRT and PTPRT activity. its synaptic substrates linked to adhesion molecules like neuroligin and neurexin by forming additional cis interactions. It has been Depression-like behaviors are modulated by PTPRT reported that knock-in mice expressing inactive PTPRT-D1046A expression in the dentate gyrus have higher social approach scores than wild-type mice (Rajamani To confirm whether deficiency of the PTPRT gene enhances et al., 2015). Recently, several studies have reported that deficits in depression-like behaviors, PTPRT was knocked down transiently, expression of the PTPRT gene induce abnormal brain functions and and depression-like behaviors were examined (Fig. 2E–G). When neurological diseases including depressive disorder; however, the lentivirus containing shRNA targeting PTPRT (Lenti-PTPRT- molecular and physiological mechanisms are not well understood shRNA) was injected into the dentate gyrus of the hippocampus, (Schuurs-Hoeijmakers et al., 2013; Quintela et al., 2017; Allen- mice displayed significantly enhanced depression-like behaviors, Brady et al., 2009; Aston et al., 2005; Hyde et al., 2016; Roberson- compared with control mice (Lenti-GFP). When the same Lenti- Nay et al., 2018 preprint). PTPRT-shRNA was injected into the CA1 region of the When we examined the behaviors of PTPRT-null and PTPRT- hippocampus, mice did not display the enhanced depression-like D401A-mutant mice, they displayed enhanced depression-like behaviors (Fig. S2). On the other hand, when adeno-associated virus behaviors compared to wild-type mice. PTPRT-deficient mice (AAV-GFP) containing the PTPRT gene (AAV-PTPRT) was injected exhibited dysregulated synaptic functions and attenuated into the dentate gyrus of PTPRT-null mice, the rescued expression of hippocampal neurogenesis. We investigated new physiological PTPRT relieved them from the depression-like behaviors effectively roles of PTPRT using a multidisciplinary approach, and suggest that (Fig. 2H–J). These results show that depression-like behaviors were PTPRT deficiency plays a crucial role in the depression-like behaviors enhanced by the PTPRT deficiency in the dentate gyrus, and that of mice. depression-like behaviors of PTPRT-null mice were ameliorated by the PTPRT rescue. RESULTS Depression-like behaviors are enhanced in PTPRT-null mice Depression-like behaviors are enhanced in mutant mice PTPRT−/− null mice were generated by targeting exon 22 of PTPRT, expressing inactive PTPRT which, as described previously, encodes the first catalytic domain PTPRT gene-targeted mutant mice carrying a missense mutation (Zhao et al., 2010). When the expression of PTPRT protein was (D401A) in the extracellular FN domain were generated by screening examined with western blotting of samples from the brains of wild- of the ENU-driven mutant mouse genomic DNA library and type (PTPRT+/+), PTPRT+/− heterozygote, and PTPRT−/− null subsequent in vitro fertilization (Sakuraba et al., 2005). Sequencing mice, PTPRT disappeared completely in the PTPRT-null mice genomic DNA of PTPRT-D401A-mutant mice confirmed the (Fig. 1A). No remarkable differences were observed in the Nissl- replacement of the highly conserved aspartic acid residue with stained brain sections of PTPRT-null mice compared with those of alanine (Fig. 3A). The expression of PTPRT protein in the brains of wild-type mice (Fig. S1). PTPRT-null mice did not display any PTPRT-D401A-mutant mice was comparable to that in wild-type aberrant phenotypes in the behavioral tests on motor functions, social mice (Fig. 3B). It has been found that PTPRT regulates the synaptic interaction, anxiety, learning and memory (Fig. 1B–L). However, formation of hippocampal neurons through the interaction between its PTPRT-null mice displayed enhanced depression-like behaviors extracellular ectodomains (Lim et al., 2009). Recombinant PTPRT compared with wild-type mice on the tail suspension test (TST), protein with the D401A mutation displayed augmented interactions forced swim test (FST) and sucrose preference test (Fig. 1M–O). between the ectodomains compared with wild-type PTPRT Intraperitoneal injection of imipramine, an antidepressant, reduced (Fig. 3C). When PTPRT expressed in heterologous cells was depression-like behaviors in PTPRT-null mice (Fig. 1P,Q). These immunoprecipitated with anti-pY912-PTPRT antibody, more results suggest that a deficiency of the PTPRT gene enhances PTPRT protein with the D401A mutation was phosphorylated depression-like behaviors in mice. compared with wild-type PTPRT (Fig. 3D). Similarly, the level of inactive phospho-PTPRT was increased in the brains of PTPRT- PTPRT expression and PTPRT activity are attenuated by D401A-mutant mice compared with that in wild-type mice chronic restraint stress (Fig. 3E). In a previous study, overexpression of PTPRT was To characterize the association between PTPRT and depression-like found to increase the synaptic formation of hippocampal neurons behaviors, the chronic restraint stress model, a well-validated model (Lim et al., 2009). When overexpressed in hippocampal neurons, of depression, was used (Chiba et al., 2012). The mice subjected to PTPRT-D401A did not increase synaptic formation as much as chronic restraint stress for 14 days displayed significantly enhanced wild-type PTPRT (Fig. 3F–H). These results show that the D401A depression-like behaviors on the TST and FST, compared with mutation attenuates PTPRT activity through aberrantly regulated control mice (Fig. 2A,B). Interestingly, the expression of PTPRT interactions between extracellular ectodomains. When behavioral was reduced in the hippocampus of the mice exposed to chronic tests were performed, PTPRT-D401A-mutant mice also displayed restraint stress compared with expression in control mice (Fig. 2C), enhanced depression-like behaviors, compared with wild-type mice whereas PTPRT expression was not significantly changed in other (Fig. 3I–K). No other aberrant phenotypes were observed in brain regions. Previously, we found that PTPRT is inactivated by the PTPRT-mutant mice (Fig. S3). Intraperitoneal injection of phosphorylation of tyrosine 912, which is located in the wedge of imipramine relieved the PTPRT-D401A-mutant mice from the the catalytic domain (Lim et al., 2009). Here, an anti-pY912-PTPRT depression-like behaviors (Fig. 3L,M). These results show that the antibody was produced to detect the phosphorylation of tyrosine depression-like behaviors observed in PTPRT-D401A-mutant mice

912, and inactive phospho-PTPRT proteins were identified in the are induced by defective PTPRT activity. Journal of Cell Science

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Decreased synaptic formation and reduced AMPA receptors in vitro at 15 days (DIV 15), puncta of GluR1 (also known as in PTPRT-deficient mice GRIA1) and GluR2 were significantly decreased in number and The numbers of excitatory and inhibitory synapses were decreased intensity compared with those in wild-type mice (Fig. 4E,F). These in the hippocampal neurons of PTPRT-null and D401A-mutant results suggest that the decrease in excitatory synapses in the mice compared with those in wild-type mice (Fig. 4A,B). Neonatal hippocampal neurons of PTPRT-deficient mice could be relevant to PTPRT-null mice (P7, postnatal day 7) showed reduced expression the reduced expression of glutamate receptors. of AMPA-type glutamate receptors, especially GluR2 (also known as GRIA2), in hippocampal tissues compared with wild-type mice Glutamate receptor trafficking is regulated by PTPRT (Fig. 4C). However, the expression of AMPA receptors was not activity altered in the hippocampus of juvenile PTPRT-null mice (P21, Immunoprecipitation with anti-PTPRT antibody was performed in the postnatal day 21) compared with wild-type mice (Fig. 4D). When rat synaptosome to characterize the interaction between PTPRT and the hippocampal neurons of PTPRT-null mice were immunostained AMPA receptors, and we observed that considerable amounts of

Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. Enhanced depression-like behaviors of PTPRT-null mice. and GluR2 were expressed in PTPRT-knockdown hippocampal − (A) Expression of PTPRT in the brains of wild-type (+/+), PTPRT+/ - neurons, and their distributions on the surface and the internal side −/− heterozygote and PTPRT -null mice examined by western blotting with anti- were analyzed. The distributional ratio of GluR1 between the surface PTPRT antibody. PTPRT decreased in PTPRT-heterozygote mice and disappeared completely in PTPRT-null mice. The two PTPRT bands represent and internal side did not change, but the distributional ratio of GluR2 full-length and endogenously cleaved forms (210 kDa and 110 kDa, was altered significantly by the knockdown of PTPRT. These results respectively; Lim et al., 2009). α-Tub, α-tubulin loading control. (B) General suggest that the membrane trafficking of GluR2 could be regulated by motor activity was conducted by open field test in wild-type (PTPRT+/+) and PTPRT in hippocampal neurons. PTPRT-null (PTPRT−/−) mice (n=11, 13). For behavioral tests, 8-week-old male mice were used. (C) Motor coordination ability was conducted by rotarod Long-term potentiation is augmented in the hippocampus test in wild-type and PTPRT-null mice (n=8, 11). (D) Rearing numbers of wild- of PTPRT-deficient mice type and PTPRT-null mice (n=10, 11) were measured in a cylinder test. (E) Self-grooming time for wild-type and PTPRT-null mice (n=10, 11) were Long-term potentiation (LTP) was measured in the hippocampus of measured in a cylinder test. (F) Sociability of wild-type and PTPRT-null mice PTPRT-deficient mice to examine whether synapse plasticity was (n=10, 11) was conducted by social interaction between two mice. changed by defective PTPRT activity. The magnitudes of LTP at the (G) Sociability of wild-type and PTPRT-null mice (n=10, 11) was conducted by perforant–dentate gyrus synapse were significantly higher in both three-chamber social interaction test. The time spent in each test chamber is PTPRT-null and D401A-mutant mice than in wild-type mice ’ indicated. **P<0.01 (Student s t-test). (H) Working memory of wild-type and (Fig. 6A,E), whereas the magnitudes of LTP at the Schaffer PTPRT-null mice (n=11, 13) was investigated by Y-maze alteration test. – PTPRT (I) Novel object recognition of wild-type and PTPRT-null mice (n=8, 11) was collateral CA1 synapse did not differ between -null and performed. **P<0.01 (Student’s t-test). (J) Spatial learning and memory of wild- D401A-mutant mice and wild-type mice (Fig. 6B,F). To determine the type and PTPRT-null mice (n=9, 9) were investigated by Morris water maze basic properties of synaptic transmission in the PTPRT-deficient mice, test. (K) A light-dark box test was used to investigate the anxiety level of wild- we examined paired-pulse facilitation (PPF) ratios at the perforant– type and PTPRT-null mice (n=8, 8). (L) An elevated plus-maze test was used to dentate gyrus synapse and the Schaffer collateral–CA1 synapse. investigate the anxiety level of wild-type and PTPRT-null mice (n=11, 11). PTPRT-null and D401A-mutant mice displayed significantly lower (M) A tail suspension test (TST) was used to investigate wild-type and PTPRT- – null mice (n =11, 13). Depression-like behaviors of PTPRT-null mice were PPF ratios at the perforant dentate gyrus synapse than wild-type mice enhanced compared with behavior of wild-type mice. **P<0.01 (Student’s t- (Fig.6C,G).ThePPFratiosattheSchaffercollateral–CA1 synapse of test). (N) A forced swim test (FST) was used to investigate wild-type and the PTPRT-null and D401A-mutant mice, however, showed no PTPRT-null mice (n=11, 13). Depression-like behaviors of PTPRT-null mice significant difference from the ratios observed in wild-type mice were enhanced compared with behavior of wild-type mice. **P<0.01 (Student’s (Fig. 6D,H). These results suggest that defective PTPRT activity t-test). (O) Sucrose preference test was used to investigate wild-type and modulated the presynaptic release of glutamate, and that synaptic PTPRT-null mice (n=21, 20). Depression-like behaviors of PTPRT-null mice – were enhanced compared with behavior of wild-type mice. **P<0.01 (Student’s plasticity was augmented at the perforant dentate gyrus synapse of t-test). (P) Intraperitoneal injection of imipramine (Imi; 20 mg/kg), an PTPRT-deficient mice. antidepressant, effectively relieved PTPRT-null mice from depression-like behaviors in the TST, compared to behavior of vehicle (Veh)-treated animals. Attenuated inhibitory synaptic transmission in the dentate Imipramine, F(1,27)=56.86, P<0.0001; genotype, F(1,27)=14.45, P=0.0007; gyrus of PTPRT-deficient mice imipramine×genotype, F(1,27)=1.11, P=0.3022; n=6, 9 in wild-type and PTPRT- Because of the altered synaptic plasticity at the perforant–dentate null mice. **P<0.01 (two-way ANOVA with Bonferroni post hoc tests). gyrus synapse, synaptic transmission was measured in the dentate (Q) Imipramine effectively relieved PTPRT-null mice from depression-like behaviors in FST, compared to the behavior of vehicle-treated animals. gyrus of PTPRT-deficient mice. The frequency of miniature inhibitory

Imipramine, F(1,25)=20.41, P=0.0001; genotype, F(1,25)=6.01, P=0.0215; postsynaptic currents (mIPSCs) was decreased in the dentate gyrus imipramine×genotype, F(1,25)=1.66, P=0.2090; n=6, 8 in wild-type and PTPRT- granule cells of both PTPRT-null and D401A-mutant mice (Fig. 7B, null mice. *P<0.05, **P<0.01 (two-way ANOVA with Bonferroni post hoc tests). D; Fig. S4), whereas miniature excitatory postsynaptic currents Data in B–Q are presented as mean±s.e.m. (mEPSCs) did not change significantly compared with those in wild- type (Fig. 7A,C). Neurotransmitter levels were then analyzed in the GluR1 and GluR2 were recruited by PTPRT (Fig. 5A). In contrast, hippocampal tissues of PTPRT-deficient mice (Fig. 7E). Although when expressed in heterologous cells, GluR2 interacted directly with serotonin and catecholamines did not show changes, GABA levels PTPRT, but GluR1 did not (Fig. 5B,C). GluR2 interacted with were significantly reduced in the hippocampus of PTPRT-null and D401A-mutant PTPRT strongly compared with wild-type PTPRT D401A-mutant mice, compared with levels in wild-type. Glutamate (Fig. 5D). Interestingly, the tyrosine residues of GluR2 were was considerably reduced in D401A-mutant mice, but not in PTPRT- effectively phosphorylated by Fyn tyrosine kinase and null mice. When hippocampal slices were immunostained with anti- dephosphorylated by PTPRT, whereas those of GluR1 were not GABA antibody, GABA intensity was observed to be decreased (Fig. 5E,F). Then, immunoprecipitation with anti-phosphotyrosine significantly in the dentate gyrus of PTPRT-null mice compared with antibody (4G10) was performed, followed by western blotting to the intensity in wild-type mice (Fig. 7F). In the hippocampal CA3 detect AMPA receptors in the synaptosomes of PTPRT-deficient mice region of PTPRT-null mice, GABA intensity was decreased compared (Fig. 5G,H). It was evident that tyrosine phosphorylation of GluR2 with that in wild-type mice, but the difference was not significant was increased in PTPRT-deficient mice compared with the level of (Fig. 7G). When the interneurons releasing GABA were examined in phosphorylation in wild-type mice. These results suggest that the the dentate gyrus of PTPRT-null mice, they showed no reduction in tyrosine residues of GluR2 were dephosphorylated by PTPRT in the cell numbers compared with those of wild-type mice (Fig. S5) (Czéh brain, and that GluR2 could be a neuronal substrate of PTPRT. A et al., 2018). The expression levels of glutamic acid decarboxylase previous study has suggested that the trafficking of GluR2 is regulated (GAD), which produces GABA, and vesicular GABA transporter by certain tyrosine phosphatases, but provided no additional (vGAT), which transports GABA, were not altered in the information regarding the specific tyrosine phosphatases that might hippocampus of PTPRT-null and D401A-mutant mice (Fig. S6). regulate GluR2 trafficking (Hayashi and Huganir, 2004; Moult et al., However, expression of GAT3 (also known as SLC6A11), a 2006). Here we examined whether the trafficking of glutamate GABAergic transporter, was significantly reduced in the receptors could be regulated by PTPRT (Fig. 5I,J). HA-tagged GluR1 hippocampal tissues of PTPRT-null and D401A-mutant mice, Journal of Cell Science

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Fig. 2. PTPRT expression is attenuated by chronic restraint stress, and depression-like behaviors are modulated by PTPRT expression. (A) The TST was conducted using 8-week-old male mice subjected to chronic restraint stress for 2 h at the same time every day for 14 days. Depression-like behaviors were enhanced in the mice subjected to stress compared with control mice (n=6, 6). *P<0.05 (Student’s t-test). (B) The FST showed that depression-like behaviors were enhanced in mice subjected to stress compared with control mice (n=6, 6). **P<0.01 (Student’s t-test). (C) PTPRT levels relative to the level of actin decreased in the hippocampus of mice subjected to chronic stress compared with levels in control mice (n=7, 7). *P<0.05 (Student’s t-test). (D) Inactive PTPRT was detected using an anti-pY912-PTPRT antibody. Inactive PTPRT increased in the hippocampus of the mice subjected to stress compared with control mice (n=7, 7). *P<0.05 (Student’s t-test). Representative western blots for the experiments described in C and D are shown below the graphs, with total PTPRT, inactive PTPRT (PTPRT-pY912) and actin loading control indicated. (E) The efficiency of different Lenti-PTPRT-shRNA treatments for the knockdown of PTPRT was examined with cultured hippocampal neurons of wild-type mice. The GFP blot shows expression of GFP encoded by the Lenti-PTPRT-shRNA vector. (F) Images showing green fluorescent protein (GFP) fluorescence in the dentate gyrus injected with Lenti-GFP and Lenti-PTPRT-shRNA. Lentiviruses were bilaterally infused into the dentate gyrus of three-week-old wild-type mice, and behavioral experiments were performed after five weeks. GCL, granule cell layer; H, hilus; ML, molecular layer. Scale bar: 500 µm. (G) Depression-like behaviors were enhanced by PTPRT knockdown in the dentate gyrus of wild-type mice. The TST and FST were performed in control mice (Lenti-GFP) and PTPRT-knockdown mice (Lenti-PTPRT-shRNA) (n=9, 10) injected with lentivirus into the dentate gyrus of wild-type mice. *P<0.05 (Student’s t-test). (H) The efficiency of different AAV-PTPRT treatments for the rescue of PTPRT was examined with cultured hippocampal neurons of PTPRT-null mice. (I) Images showing GFP fluorescence in the dentate gyrus injected with AAV-GFP or AAV-PTPRT-GFP. Adeno- associated virus (AAV) was bilaterally infused into the dentate gyrus. Scale bar: 500 µm. (J) PTPRT-null mice were relieved from depression-like behaviors by rescued PTPRT expression in the dentate gyrus. The TST and FST were performed in control mice (AAV-GFP) and PTPRT-rescued mice (AAV-PTPRT) (n=8, 8) injected with AAV into the dentate gyrus of PTPRT-null mice. *P<0.05, **P<0.01 (Student’s t-test). Data in A–D, G, J are mean±s.e.m. whereas the expression of GAT1 (also known as SLC6A1) was GAT3 pulled down less D401A-mutant PTPRT than wild-type comparable to that in wild-type mice (Fig. 7H,I). GAT3 interacted PTPRT when immunoprecipitation was performed with the lysate of with PTPRT in heterologous cells as well as in the rat synaptosome heterologous cells (Fig. 7L). Interestingly, tyrosine phosphorylation of (Fig. 7J,K). Immunostaining of hippocampal slices displayed the GAT3 was increased in the synaptosome of PTPRT-null mice colocalization of PTPRT and GAT3 in the dentate gyrus (Fig. S7). compared with that in wild-type mice (Fig. 7M). These results suggest Journal of Cell Science

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Fig. 3. Enhanced depression-like behaviors of PTPRT D401A mutant mice. (A) Result of sequencing genomic DNA of PTPRT D401A-mutant mice. Diagram shows the region of the mutation introduced. Representative chromatograms from wild-type (WT), heterozygous (Het)- and homozygous (Ho)-mutant mice are shown, with the mutated sequence highlighted by the dashed box. (B) Expression of PTPRT protein in the brain of PTPRT-D401A-mutant (401MT) mice is comparable to wild-type (WT) mice. α-Tub, α-tubulin loading control. (C) The interactions between the ectodomains of PTPRT with the D401A mutation (RT-Fc- 401) were augmented compared with those of wild-type PTPRT (RT-Fc-WT) (Lim et al., 2009). Input, 2%. Quantification (immunoprecipitated protein as a percentage of input protein): trans wild-type, 38.1; trans mutated, 48.1; trans+cis wild-type, 8.3; trans+cis mutated, 8.4; cis wild-type, 260.9; cis mutated, 478.4. (D) Immunoprecipitation with anti-pY912 antibody showed that the inactive phospho-PTPRT was increased in D401A-PTPRT (401) compared with wild-type PTPRT (WT). Input, 3%. Quantification: PTPRT wild-type, 123.3; PTPRT D401A, 141.6 (immunoprecipitated protein as a percentage of input protein); PTPRT- pY912 wild-type, 26763; PTPRT-pY912 D401A, 26611 [AU (arbitrary units), integrated intensity]. (E) When immunoprecipitation was performed with anti-pY912 antibody, the inactive phospho-PTPRT was increased in the brain of D401A-mutant mice compared with the level in wild-type mice. Input, 5%. Quantification; PTPRT wild-type mice, 506.8; PTPRT D401A-mutant mice, 499.2 (immunoprecipitated protein as a percentage of input protein); PTPRT-pY912 wild-type mice, 18757; PTPRT-pY912 D401A-mutant mice, 24439 (AU, integrated intensity). (F) D401A-mutant PTPRT did not increase the density of dendritic spines compared with wild-type PTPRT when expressed in cultured hippocampal neurons. Hippocampal neurons of DIV 8 were transfected with EGFP only (Control, Ctr), or with EGFP and wild-type (RT-WT) or D401A (RT-401) PTPRT, and immunostained for EGFP after 9 days. Scale bars: 10 µm. Dendritic spines were quantified in control neurons and neurons expressing wild-type PTPRT or mutant PTPRT (n=30, 29, 29 dendrites, respectively). Tukey post hoc test after application of one- way ANOVA, ***P<0.001; F=14.84, P<0.0001. (G) D401A-mutant PTPRT did not increase the number of excitatory synapses (vGLUT-positive PSD-95 clusters) compared with wild-type PTPRT when expressed in cultured hippocampal neurons. Hippocampal neurons were immunostained for EGFP (green), PSD-95 (red), and vGLUT (blue). Scale bars: 10 µm. Excitatory synapses were quantified in control neurons and neurons expressing wild-type PTPRT or mutant PTPRT (n=30, 29, 29 dendrites, respectively). By Tukey post hoc test after application of one-way ANOVA, *P<0.05 and ***P<0.001; F=10.69, P<0.0001. (H) D401A-mutant PTPRT did not increase the number of inhibitory synapses (vGAT-positive gephyrin clusters) compared with wild-type PTPRT when expressed in cultured hippocampal neurons. EGFP (green), gephyrin (red) and vGAT (blue) images are shown. Scale bars: 10 µm. Inhibitory synapses were quantified in control neurons and neurons expressing wild-type PTPRT or mutant PTPRT (n=27, 29, 31 dendrites, respectively). By Tukey post hoc test after application of one-way ANOVA, ***P<0.001; F=11.46, P<0.0001. (I) Enhanced depression-like behaviors of D401A-mutant mice (PTPRT401MT) compared with wild-type mice (PTPRTWT) in the TST (n=9, 9). *P<0.05 (Student’s t-test). (J) Enhanced depression-like behaviors of D401A-mutant mice compared with wild-type mice in the FST (n=10, 11). **P<0.01 (Student’s t-test). (K) Enhanced depression-like behaviors of D401A-mutant mice compared with wild-type mice in the sucrose preference test (n=13, 10). **P<0.01 (Student’s t-test). (L) Intraperitoneal injection of imipramine (Imi; 20 mg/kg) effectively relieved D401A-mutant mice from depression-like behaviors relative to vehicle treatment (Veh) in the TST. Imipramine, F(1,31)=101.66, P<0.0001; genotype, F(1,31)=2.40, P=0.1318; imipramine×genotype, F(1,31)=3.28, P=0.0800; n=7, 10 in wild-type and D401A-mutant mice, respectively. **P<0.01 (two-way ANOVA with Bonferroni post hoc tests). (M) Imipramine effectively relieved D401A-mutant mice from depression-like behaviors in the FST. Imipramine, F(1,34)=10.62, P<0.05; genotype, F(1,34)=3.80, P=0.059; imipramine×genotype, F(1,34)=1.041, P=0.32; n=10, 9 in wild-type and D401A-mutant mice, respectively. *P<0.05 (two-way ANOVA with Bonferroni post hoc tests). Data in F–M are mean±s.e.m. that the synaptic function of GAT3 associated with the release applied, the inhibitory transmissions in the dentate gyrus granule cells of GABA could be regulated by PTPRT through tyrosine of PTPRT-null mice were rescued to a level comparable to that in wild- dephosphorylation. When allopregnanolone (3α,5α- type mice (Fig. 7N). In addition, the depression-like behaviors of tetrahydroprogesterone, also known as 3α,5α-THP), a potent PTPRT-null mice were ameliorated by intraperitoneal injection of positive allosteric modulator of the action of GABAA receptor, was allopregnanolone (Fig. 7O). These results indicate that attenuated Journal of Cell Science

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Fig. 4. See next page for legend. Journal of Cell Science

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Fig. 4. Attenuated synaptic formation and reduced AMPA receptors in the compared with the number differentiated from wild-type NSCs hippocampus of PTPRT-deficient mice. (A) The numbers of excitatory (Fig. 8E). However, the number of GFAP-positive astrocytes (vGLUT-positive PSD-95 clusters, top) and inhibitory (vGAT-positive gephyrin differentiated from PTPRT-null NSCs displayed no difference from clusters, bottom) synapses were significantly decreased in hippocampal PTPRT neurons of PTPRT-null mice compared with wild-type mice. Excitatory wild type. In addition, the neurons derived from -null NSCs synapses (n=28, 28 dendrites in wild-type and PTPRT-null mice, respectively) showed reduced neurite length and attenuated dendritic arborization and inhibitory synapses (n=29, 28 dendrites in wild-type and PTPRT-null mice, compared with those derived from wild-type NSCs (Fig. 8F). These respectively) were quantified. **P<0.01, ***P<0.001 (Student’s t-test). (B) The results suggest that defective PTPRT activity attenuates the numbers of synapses were significantly decreased in hippocampal neurons proliferation and the neurogenic ability of embryonic NSCs in the 401MT prepared from D401A-mutant mice (PTPRT ) compared to wild-type mice hippocampus of PTPRT-deficient mice. (PTPRTWT). Excitatory synapses (top, n=28, 28 dendrites in wild-type and D401A-mutant mice, respectively) and inhibitory synapses (bottom, n=31, 30 Brain-derived neurotrophic factor (BDNF) plays significant roles dendrites in wild-type and D401A-mutant mice, respectively) were quantified. in neurogenesis and promotes the differentiation and proliferation of **P<0.01, ***P<0.001 (Student’s t-test). (C) Western blotting with hippocampal NSCs through activation of Akt (Bartkowska et al., 2007; Bath et al., tissues of postnatal day 7 (P7) mice displayed that the expression of GluR2 2012; Goncalves et al., 2016). In the brain synaptosome, PTPRT decreased in PTPRT-null mice compared with that in wild-type mice, whereas interacted with BDNF receptor Ntrk2 as well as with p75NTR GluR1 did not decrease significantly. Expression was quantified relative to the (NGFR), PIK3CA, PDPK1 and Akt1, which are involved in the actin loading control. GluR1 (n=10, 12) and GluR2 (n=17, 18) in wild-type and neurotrophin signal pathway (Fig. 8G). When PTPRT and Ntrk2 were PTPRT-null mice, respectively. **P<0.01 (Student’s t-test). (D) The differences of GluR1 and GluR2 expression between wild-type and PTPRT-null mice were expressed in heterologous cells, PTPRT was pulled down effectively not significant in the hippocampal tissues of postnatal day 21 (P21) mice. by Ntrk2 (Fig. 8H). Interestingly, the phosphorylation level of Expression was quantified relative to the actin loading control. GluR1 (n=7, 7) activity-dead PTPRT (PTPRT-2CS) was higher than that of wild- and GluR2 (n=7, 7) in wild-type and PTPRT-null mice, respectively. type PTPRT (PTPRT-WT) because of auto-dephosphorylation (E) Immunostaining with anti-GluR1 antibody showed that the number and (Fig. 8I) (Lim et al., 2009). These results suggest that PTPRT can intensity of GluR1 puncta decreased in hippocampal neurons (15 days in vitro, interact with Ntrk2 in the brain, and that the tyrosine residues DIV) of PTPRT-null mice compared with those of wild-type mice. Boxes indicate regions shown in magnified images on the right. Number of GluR1 of PTPRT are phosphorylated by Ntrk2 tyrosine kinase. The puncta and GluR1 intensity (A.U, arbitrary intensity units) were quantified in phosphorylations of Akt1 at Ser124 and PDPK1 at Ser241 were wild-type and PTPRT-null mice (n=31, 26 dendrites, respectively). *P<0.05, decreased significantly in the hippocampal tissues of PTPRT-null ***P<0.001 (Student’s t-test). (F) The number and intensity of GluR2 puncta mice compared with their phosphorylation in wild-type mice (Fig. 8J, decreased in hippocampal neurons of PTPRT-null mice compared with those K). PTPRT-null NSCs also showed decreased phosphorylation of of wild-type mice. Boxes indicate regions shown in magnified images on the Akt1 at Ser473 and S6 at Ser240/244 compared with levels in wild- right. Number of GluR2 puncta and GluR2 intensity were quantified in wild-type type NSCs (Fig. 8L). The expression of Ntrk2 was not altered in the and PTPRT-null mice (n=31, 26 dendrites, respectively). *P<0.05, ***P<0.001 PTPRT (Student’s t-test). Data in A–F are presented as mean±s.e.m. Scale bars in A, hippocampal tissue or the NSCs of -null mice compared with B,E,F: 10 µm. wild type (Fig. S8). Although the regulation mechanism is not clear so far, PTPRT appears to play pivotal roles in neurogenesis in the dentate gyrus and regulation of the proliferation and differentiation of GABAergic synaptic function of the dentate gyrus granule cells could hippocampal NSCs. be associated with the depression-like behaviors of PTPRT-deficient mice. DISCUSSION In this study, we found that defective PTPRT activity induced Delayed development of newborn cells in PTPRT-deficient depression-like behaviors in PTPRT-null and D401A-mutant mice mice through a dysregulation in synaptic functions and aberrant Attenuated neurogenesis has been shown in the dentate gyrus of hippocampal neurogenesis. PTPRT regulates the synaptic formation patients with depression (Boldrini et al., 2009; Lucassen et al., 2010; as well as the dendritic arborization of hippocampal neurons (Lim Boldrini et al., 2013; Yun et al., 2016). When hippocampal et al., 2009; Park et al., 2012; Lee, 2015). Several genetic studies have neurogenesis was investigated in PTPRT-deficient mice after suggested that deficits of the PTPRT gene induce abnormal brain intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU) functions and neurological diseases. Cases of a point mutation and followed by brain sampling 2 days later, there was no difference in copy number gain of the PTPRT gene were suspected as pathogenic the number of BrdU-positive dividing nerve cells in the dentate gyrus in a family and a child with intellectual disability, respectively of PTPRT-null and wild-type mice (Fig. 8A). However, the number (Schuurs-Hoeijmakers et al., 2013; Quintela et al., 2017). In addition, of newborn granule cells expressing doublecortin (DCX) was a genome-wide linkage analysis in a six-generation family has reduced, and the neurite length of the cells decreased in the dentate mapped the PTPRT gene within a region significant for autism gyrus of PTPRT-null and D401A-mutant mice (Fig. 8B,C). These spectrum disorder (ASD) (Allen-Brady et al., 2009). On the other results show that the defective PTPRT activity attenuated hand, a clinical case study has reported that patients with depression neurogenesis as well as the maturation of newborn granule cells in display altered expression of the PTPRT gene in the temporal cortex the dentate gyrus of PTPRT-deficient mice. The proliferation and (Aston et al., 2005). A GWAS analysis with a large sample size of multi-lineage differentiation of neural stem cells (NSCs) were then patients with depression revealed that the PTPRT gene is included in examined in cultured embryonic NSCs prepared from the the loci associated with a risk of major depression (Hyde et al., 2016). hippocampus of PTPRT-null mice. The results showed a decrease A recent study analyzed patients with early-onset major depression in the number of PTPRT-null NSCs derived from secondary and reported that the PTPRT gene is included in regions of DNA neurospheres compared with the number of wild-type NSCs methylation enrichment (Roberson-Nay et al., 2018 preprint). derived in the same manner (Fig. 8D). When neurogenic ability PTPRT-null and D401A-mutant mice in this study displayed was examined by counting the number of Tuj1-positive neurons normal phenotypes on behavioral tests of social interaction, anxiety, (indicating expression of neuron-specific β-tubulin) differentiated learning and memory, but had enhanced depression-like behaviors from PTPRT-null NSCs, a significant reduction was observed (Figs 1, 3). In the hippocampus of mice subjected to chronic stress, a Journal of Cell Science

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Fig. 5. See next page for legend. Journal of Cell Science

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Fig. 5. Glutamate receptor trafficking is regulated by PTPRT activity. (A) substrate of PTPRT in the brain, and that the expression of GluR2 is Interaction between AMPA glutamate receptors and PTPRT in the brain. When regulated by PTPRT. A previous study has suggested that the immunoprecipitation was performed with rat synaptosome, GluR2 and GluR1 trafficking of GluR2 is regulated by tyrosine phosphatases, but the were pulled down by anti-PTPRT antibody (IP:RT). Input, 5%. Quantification: GluR2, 61.1; GluR1, 122.2; PTPRT, 470.7 (immunoprecipitated protein as a authors did not specify which tyrosine phosphatases might be percentage of input protein). (B) When PTPRT and GluR1 were expressed in involved in this (Hayashi and Huganir, 2004; Moult et al., 2006). The heterologous cells, anti-PTPRT (left) and anti-GluR1 antibodies (right) were distribution of GluR2 between the surface and internal side was used to pull down GluR1 and PTPRT, respectively. GluR1 was not pulled down altered by PTPRT knockdown, and PTPRT could regulate the by anti-PTPRT antibody, and vice versa. Input, 3%. Trf, transfection. (C) When membrane trafficking of GluR2 (Fig. 5I,J). Defective PTPRT activity expressed in heterologous cells, GluR2 and PTPRT were pulled down by anti- induced dysfunctions of AMPA receptors and attenuated the PTPRT (left) and anti-GluR2 (right) antibodies, respectively. Input, 3%. formation of excitatory synapses in PTPRT-deficient mice. It has (D) GluR2 pulled down D401A mutant PTPRT (RT-401) more strongly than wild- GluR2 type PTPRT (RT-WT) when immunoprecipitation was performed in the lysate of been shown previously that -null mice exhibit enhanced LTP heterologous cells transfected with GluR2 and RT-WT or RT-401. Input, 3%. similar to PTPRT-deficient mice (Jia et al., 1996), and here we found Quantification: 1.48, RT-401/RT-WT immunoprecipitated by GluR2. (E) When that dysregulated GluR2 in PTPRT-deficient mice augmented expressed in heterologous cells, GluR1 was not phosphorylated by Fyn tyrosine LTP (Fig. 6A,E). Although PTPRT-deficient mice exhibited kinase nor dephosphorylated by PTPRT. Fyn-CA, constitutively active Fyn; Fyn- altered hippocampal synaptic plasticity, they did not display KD, the kinase-dead Fyn; PTPRT-YF, constitutively active PTPRT. Input, 3%. reduced learning and memory in the behavioral tests (Fig. 1H–J; (F) GluR2 was phosphorylated by Fyn tyrosine kinase and dephosphorylated – effectively by PTPRT in heterologous cells. Input, 3%. Quantification: pY-GluR2 Fig. S3N Q). A previous study reported that juvenile rats exposed to with Fyn-CA, 283.7; pY-GluR2 with Fyn-CA and PTPRT-YF, 47.4 chronic stress have impaired recognition memory through the reduced (immunoprecipitated protein as a percentage of input protein). (G) Tyrosine expression of glutamate receptors and attenuated glutamatergic phosphorylation of GluR2 was increased in the synaptosome of PTPRT-null transmission in prefrontal cortex (PFC), but not in hippocampus mice (−/−) compared with that of wild-type mice (+/+). Immunoprecipitation (Yuen et al., 2012). The authors suggested that PFC has higher using anti-phosphotyrosine antibody (4G10) was performed with synaptosomes sensitivity to chronic stress than hippocampus regarding cognitive of PTPRT-null followed by western blotting. 4G10, anti-phosphotyrosine antibody; 4G10+pY, anti-phosphotyrosine antibody pre-incubated with O- impairment. We observed that PTPRT expression was changed in phospho-L-tyrosine. Data are mean±s.e.m ratios of tyrosine phosphorylation in hippocampus by chronic stress, but was not significantly changed in PTPRT-null mice to tyrosine phosphorylation in wild-type mice (n=25 and 13 for cortex (Fig. 2C). Our results suggest that the alteration of PTPRT-null and wild-type mice, respectively). *P<0.05 (Student’s t-test). (H) glutamatergic function in the hippocampus of PTPRT-deficient Immunoprecipitation using anti-phosphotyrosine antibody was performed with mice could induce depressive behaviors, but has little effect on synaptosomes of D401A-mutant mice (MT) and wild-type mice (WT) followed by working memory. western blotting for the indicated proteins. Data are mean±s.e.m. ratios of tyrosine phosphorylation in D401A-mutant mice to tyrosine phosphorylation in The transient knockdown of PTPRT in the dentate gyrus induced wild-type mice (n=12 and 6 for mutant and wild-type mice, respectively). depression-like behaviors, but PTPRT knockdown in the CA1 region Tyrosine phosphorylation of GluR2 was increased in the D401A-mutant mice did not (Fig. 2F,G; Fig. S2). Augmented LTP was observed at the compared with wild-type mice, but the difference was not significant. (I) When perforant–dentate gyrus synapse, but not at the Schaffer collateral– HA–GluR1 was expressed in hippocampal neurons, the distributional ratio of CA1 synapse, of PTPRT-deficient mice (Fig. 6). Inhibitory synaptic GluR1 between surface and the internal side was analyzed. The distribution of transmission was decreased in the dentate gyrus granule cells of GluR1 was not changed by the knockdown of PTPRT compared with control. PTPRT-deficient mice (Fig. 7B,D). The dentate gyrus is well known Mean±s.e.m. GluR1 intensity (A.U, arbitrary intensity units) and distribution ratio are shown. n=8 and 7 for control and PTPRT knockdown, respectively. *P<0.05 for generating new neurons during adulthood. Here, PTPRT-deficient (Student’s t-test). (J) The distribution of GluR2 between surface and the internal mice displayed aberrant development of newborn granule cells in side was changed significantly by the knockdown of PTPRT compared with their dentate gyrus (Fig. 8B,C). Adult hippocampal neurogenesis has control. Mean±s.e.m. GluR2 intensity and distribution ratio are shown. n=7 for been evaluated as a candidate mechanism for the etiology of both control and PTPRT knockdown. **P<0.01, ***P<0.001 (Student’s t-test). depressive disorder (Yun et al., 2016; Sheline et al., 1996; Bremner Scale bars in I and J: 50 µm. et al., 2000; Geuze et al., 2005). In an earlier study using an animal model of depression, hippocampal neurogenesis was found to buffer stress responses and ameliorate depressive behaviors (Snyder et al., well-validated model of depression, PTPRT expression was reduced 2011). Most antidepressants and antidepressant-like behavioral and inactive phospho-PTPRT expression increased (Fig. 2C,D). effects stimulate adult hippocampal neurogenesis (Boldrini et al., Basic and clinical studies have shown that depressive disorder is 2009; Sahay and Hen, 2007; David et al., 2009). It has also been associated with attenuated neuronal synaptogenesis (Duman and suggested that adult neurogenesis could play a role in the structural Aghajanian, 2012). Here, hippocampal neurons of PTPRT-deficient plasticity of the mature brain by generating and adding newborn mice displayed a decrease in excitatory and inhibitory synapses neurons into the existing brain circuitry (Eliwa et al., 2017; Bond (Fig. 4A,B). NMDA receptor antagonists have been suggested to et al., 2015). In a recent study, it was observed that young adult-born exert rapid antidepressant effects by enhancing AMPA receptor granule cells in the dentate gyrus activate local GABAergic functions, and the US FDA have approved esketamine [S(+) interneurons to evoke strong inhibitory input into mature granule enantiomer of ketamine] for patients with treatment-resistant cells (Drew et al., 2016), suggesting that attenuated inhibitory depression (Maeng et al., 2008; Jaso et al., 2017). We found that synaptic transmission in the granule cells of PTPRT-deficient mice neonatal PTPRT-null mice exhibited reduced expression levels of could be induced by aberrant neurogenesis in the dentate gyrus AMPA receptor GluR2, compared with levels in wild-type mice (Fig. 7B,D). It has been reported that signal pathways of neurotrophic (Fig. 4C). Hippocampal neurons prepared from PTPRT-null mice factors control hippocampal neurogenesis and neuronal processes also displayed decreased AMPA receptor levels (Fig. 4E,F). PTPRT (Krishnan and Nestler, 2008). Clinical studies have shown that serum effectively interacted with GluR2 and dephosphorylated tyrosine levels of BDNF are decreased in major depressive patients, and the residues of GluR2 (Fig. 5C,F), and tyrosine phosphorylation of administration of BDNF produces antidepressant-like activity GluR2 was increased in the brains of PTPRT-deficient mice (Fig. 5G, through the modification of neurogenesis in an animal model of

H). These results suggest that GluR2 could be an endogenous depression (Hashimoto et al., 2004; Siuciak et al., 1997; Vithlani Journal of Cell Science

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Fig. 6. Augmented long-term potentiation in PTPRT-deficient mice. (A) LTP was augmented at the perforant–dentate gyrus (DG) synapse of PTPRT-null mice compared with that of wild-type mice. Left panel shows averaged timecourse changes in field excitatory post-synaptic potential (fEPSP) slope induced by theta burst stimulation (TBS). Bic, bicuculline. Right panel shows LTP as a percentage of baseline at 60 min post TBS. Mean±s.e.m. *P<0.05 (Student’s t-test). (B) LTP was not changed at the Schaffer collateral–CA1 synapse of PTPRT-null mice compared with that of wild-type mice. Left panel shows averaged timecourse changes in fEPSP slope induced by TBS. Right panel shows LTP as a percentage of baseline at 60 min post TBS. Mean±s.e.m. (C) Paired-pulse facilitation (PPF) ratio was reduced at the perforant–DG synapse of PTPRT-null mice compared with that of wild-type mice. Mean±s.e.m. *P<0.05 (Student’s t-test). (D) PPF ratio was not changed reduced at the Schaffer collateral–CA1 synapse of PTPRT-null mice compared with that of wild-type mice. Mean±s.e.m. (E) LTP was augmented at the perforant–DG synapse of D401A-mutant mice compared with that of wild-type mice. Left panel shows averaged timecourse changes in fEPSP slope induced by TBS. Right panel shows LTP as a percentage of baseline at 60 min post TBS. Mean±s.e.m. *P<0.05 (Student’s t-test). (F) LTP was not changed at the Schaffer collateral–CA1 synapse of D401A-mutant mice compared with that of wild-type mice. Left panel shows averaged timecourse changes in fEPSP slope induced by TBS. Right panel shows LTP as a percentage of baseline at 60 min post TBS. Mean±s.e.m. (G) PPF ratio was reduced at the perforant–DG synapse of D401A-mutant mice compared with that of wild-type mice. Mean±s.e.m. *P<0.05 (Student’s t-test). (H) PPF ratio was not changed reduced at the Schaffer collateral–CA1 synapse of D401A-mutant mice compared with that of wild-type mice. Mean±s.e.m. In A–C and E–G, numbers in parentheses are the numbers of animals and slices tested. Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243972. doi:10.1242/jcs.243972 et al., 2013). Chronic treatment with antidepressants increases BDNF (Permit Number: KRIBB-AEC-17152) and the Institutional Animal Care expression and upregulates BDNF signaling that promotes adaptive and Use Committee of Eulji University (Permit Number: EUIACUC 11-12). neuronal plasticity (Nibuya et al., 1995; Russo-Neustadt et al., 2000; Alme et al., 2007). Here, PTPRT interacted with BDNF receptor Mice PTPRT−/− Ntrk2 and signaling molecules in the BDNF pathway (Fig. 8G). In null mice were generated by targeting exon 22 of PTPRT, which PTPRT encodes the first catalytic domain, as described previously (Zhao et al., the hippocampal tissues and NSCs of -null mice, the 2010). PTPRT-D401A-mutant mice were generated by screening the phosphorylation of Akt1, PDPK1 and S6 was attenuated N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library (Fig. 8J–L). It has been shown previously that the activation of and subsequent in vitro fertilization (Sakuraba et al., 2005). Since then, Akt1 by phosphoinositide 3-kinase (PI3K) promotes the PTPRT-null and D401A-mutant mice were backcrossed for more than 10 differentiation of NSCs into GABAergic neurons (Chang et al., generations onto the C57BL/6J genetic background. Animals were housed 2016; Oishi et al., 2009). Hippocampal NSCs of PTPRT-null mice in a temperature-controlled (24°C) environment under a 12-h light/dark displayed aberrant proliferation and multi-lineage differentiation cycle (lights on at 7 am) with food and water ad libitum. (Fig. 8D,E). Attenuated hippocampal neurogenesis could thus induce dysregulation of inhibitory synapses in PTPRT-deficient mice. Behavioral tests Many clinical studies have reported that GABA concentrations are 8-week-old male mice were used for behavioral tests. reduced in patients with depression, compared with concentrations in Forced swim test controls ( Luscher et al., 2011; Gabbay et al., 2012; Smiley et al., 2016; The forced swim test (FST) was performed as previously described (Kim Romeo et al., 2018). On the other hand, it has been shown that the and Han, 2006). Briefly, each mouse was habituated to the test room and expression and function of GAD, the crucial enzyme producing GABA, were placed in a transparent Plexiglas cylinder containing water for 6 min. is not changed in patients with depression (Pehrson and Sanchez, 2015). The immobility time during the last 5 min was recorded and scored by a In the current study, PTPRT-deficient mice also displayed reduced blinded investigator. hippocampal GABA concentrations, but not altered expressions of GAD and vGAT GABA transporter (Fig. 7E–G; Fig. S6). The Tail suspension test expression of GAT3 GABA transporter was significantly decreased The tail suspension test (TST) was performed as previously described (Kim in the hippocampus of PTPRT-deficient mice (Fig. 7H). Earlier and Han, 2006). Briefly, after habituation, each mouse was suspended by the studies have shown that GAT3 is reduced in helpless-rat models of tail using adhesive tape for 6 min. The immobility time during the last 5 min depression, whereas the expression levels of GAT1, GAD and vGAT was recorded and scored by a blinded investigator. do not change (Zink et al., 2009). PTPRT interacted with GAT3, and tyrosine phosphorylation of GAT3 was increased in PTPRT-null Sucrose preference test – Mice were habituated to sucrose over a 3 day period by replacing water mice (Fig. 7J M). The expression of GAT3 appeared to be regulated bottles with bottles containing sucrose solution (1%). Mice were given free by PTPRT in a manner similar to GAT1, whose function is regulated access to two bottles, one with tap water and the other with 1% sucrose through tyrosine dephosphorylation (Whitworth and Quick, 2001). In solution, after water deprivation of 16 h. After 1 h, the weights of bottles another recent study, the disinhibition of somatostatin-positive were measured for calculation of fluid consumption. Sucrose preference was GABAergic interneurons was seen to alleviate the depressive obtained by calculating the sucrose intake as a percentage of total intake: behaviors of an animal model of depression (Fuchs et al., 2017). sucrose intake 100. Many studies have suggested that enhancing GABAergic inhibitory sucrose intake þ water intake synaptic inputs could be an effective strategy for antidepressant Open field test therapy. Recently, the US FDA announced allopregnanolone (also Locomotor activity was assessed by measuring the distance of movement in known as brexanolone), a potent positive allosteric modulator of an open field that was made of white Plexiglas (45×45×40 cm). Each mouse GABAA receptors, as the first-ever approved drug to treat postpartum was habituated the test room for ∼30 min. After that, the mice were placed depression (Kanes et al., 2017; Gee et al., 1995; Lambert et al., 2003; individually in the arena for 60 min. The horizontal locomotion of mice in Reddy, 2010). Here, treatment with allopregnanolone rescued an open field was measured using a computerized video tracking system, inhibitory synaptic transmission and ameliorated the depression-like SMART (Panlab, Barcelona, Spain). behaviors of PTPRT-deficient mice (Fig. 7N,O). These results suggest that the attenuation of GABAergic inhibitory synaptic functions Rotarod test induced the depression-like behaviors of PTPRT-deficient mice. The rotarod test was performed as follows: for evaluation of coordination and motor learning the accelerating rotarod (Daejong Instrument Industry In conclusion, we observed enhanced depression-like behaviors Co., Seoul, Korea) test was performed on three consecutive days. The mice in PTPRT-deficient mice and examined the physiological roles of were given three trials a day with an intertrial interval of 30 min. PTPRT that might be related to depressive disorder. PTPRT Acceleration speed from 4 to 40 rpm over a 5 min period was chosen. regulated the expression of AMPA receptors as well as membrane The latency to fall off was the measure of motor coordination and trafficking of GluR2 and, in addition, controlled GABAergic improvement across trials was the measure of motor learning. The cut-off synaptic functions and neurogenesis in the dentate gyrus. Our time was set at 5 min. results suggest that PTPRT deficiency could play a crucial role in the etiology of depressive disorder, and that PTPRT-deficient mice Cylinder test should be used for understanding the physiological mechanisms The cylinder test was performed as previously described (Park et al., 2016). underlying depressive disorder. Sensorimotor function was evaluated using the cylinder test. Each mouse was placed into a transparent acrylic cylinder (diameter, 20 cm) and recorded for 5 min. The number of times that the right and left forelimbs MATERIALS AND METHODS came into contact with the wall were counted by a blinded observer. Ethical statement This study was performed in accordance with the relevant guidelines under Light-dark box test Korean law. All animal use was approved by the Animal Use and Care The light-dark box test was performed as follows: the light box

Committee of Korea Research Institute of Bioscience and Biotechnology (20×20×20 cm) and dark box (10×20×20 cm) were made of white Journal of Cell Science

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Fig. 7. See next page for legend.

Foamex, with a shuttle door (5×7 cm) between the two at floor level. The was presented in 10-ml serological pipettes with sippers attached with light box was open at the top and illuminated with an 800 lux light at parafilm. Pipettes were closed with rubber stoppers and positioned through the bottom. The dark box was painted black and had a removable black lid. wire cage lids. In home-cage testing, the latency to drink and the volume The test started by placing a mouse into the dark box, with the shuttle door consumed were recorded every 5 min for 30 min. Home-cage testing closed initially. After 10 s, the shuttle door was opened. The total numbers occurred under dim lighting (∼50 lx). Then, in novel-cage testing, mice of transitions between the dark and light compartments, crossings, and the were placed into new clean cages of the same dimensions but without time spent in the light box during a 5-min period were measured. shavings, with pipettes containing the milk positioned. Novel-cage testing occurred under bright lighting (∼1200 lx), with white paper placed under Elevated plus-maze test cages to enhance aversiveness. The elevated plus-maze (EPM) test was performed as follows: the EPM was made of gray Formax. The apparatus consisted of four arms (30×7 cm), Novel object recognition test which were elevated 50 cm above the floor and placed at right angles to each Novel object recognition is a validated and widely used test for assessing other. Two of the arms had 20-cm high walls (closed arms), while the other recognition memory (Park et al., 2017). Mice were placed individually in a two had no walls (open arms). Each mouse was placed at the center of the 40×20×20 cm testing chamber for 10 min with two identical objects platform and left to explore the arms for 5 min. The number of entries into (familiar, acquisition session). Mice were returned to home cages and placed the open and enclosed arms and the time spent in each arm and center area back into the testing chamber 24 h later, in the presence of one of the original were recorded. Entry into each arm was scored as an event if the animal objects and one novel object (novel, recognition session) for 10 min. The placed all paws into the corresponding arm. original objects consisted of cylindrical wooden blocks, which had a height and diameter of 10 cm and 2 cm, respectively. The novel object was a Hyponeophagia test rectangular wooden block that was 10×2.5×2 cm. The acquisition and Hyponeophagia refers to the inhibition of feeding produced by exposure to recognition sessions were video recorded and a blinded observer scored the novelty. Mice were trained to drink sweetened condensed milk for three time spent exploring the objects and the number of times the objects were consecutive days. Mice were presented with diluted (1:3 ratio of milk:water) touched. Exploration was defined as sniffing and touching the object with sweetened condensed milk (Carnation, NC, USA) for 30 min each day. Milk the nose and/or forepaws. The duration and number of times spent exploring Journal of Cell Science

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Fig. 7. Attenuated inhibitory synaptic function in PTPRT-deficient mice. Y-maze alternation test (A) Excitatory synaptic transmissions were examined in granule cells of The Y-maze alternation test was conducted as previously described (Park dentate gyrus, and mEPSC was not altered in PTPRT-null mice compared with et al., 2017). A Y-shaped maze with three transparent arms of 50×10×12 cm wild-type mice. n=12 and 13 in wild-type and PTPRT-null mice, respectively. was used. Each mouse was placed at the end of one arm facing the center and The analysis of sEPSC and sIPSC is shown in Fig. S4. (B) Inhibitory synaptic allowed to explore the maze for a period of 10 min. The sessions were video transmissions were examined in granule cells. The frequency of mIPSC was recorded and scored for entries into arms. The percentage of spontaneous decreased significantly in PTPRT-null mice compared with that in wild-type alternation was calculated as the ratio of the actual to possible alternations mice. n=10 and 15 in wild-type and PTPRT-null mice, respectively. *P<0.05 (defined as the total number of arm entries minus 2), which was multiplied (Student’s t-test). (C) mEPSC was not altered in granule cells of D401A-mutant by 100(Conrad et al., 1996): mice (PTPRT401MT) compared with that in granule cells of wild-type mice (PTPRTWT). n=12 and 17 in wild-type and D401A-mutant mice, respectively. number of alternations alternation (%) ¼ 100. (D) The frequency of mIPSC decreased significantly in D401A-mutant mice (total arm entries 2Þ compared with wild-type mice. n=14 in both wild-type and D401A-mutant mice. **P<0.01 (Student’s t-test). (E) The amount of GABA was reduced significantly Social interaction test in hippocampal tissues of PTPRT-null and D401A-mutant mice when analyzed The social interaction test was conducted as previously described (Felix- with an amino acid analyzer. n=5 for both wild-type and PTPRT-null mice; n=6 Ortiz and Tye, 2014). The social interaction chamber consisted of white and 5 for wild-type and D401A-mutant mice, respectively. Glutamate was acryl-walled box (40×20×20 cm). A single mouse was placed in the test box reduced in in D401A-mutant mice. *P<0.05 (Student’s t-test). (F) GABA for 3 min for habituation. After that, a novel male mouse was introduced to intensity was decreased significantly in the dentate gyrus of PTPRT-null mice the test cage and allowed to explore freely for 3 min. All behaviors were compared with wild-type mice when immunostained with anti-GABA antibody. video recorded, and social interaction, such as body sniffing, anogenital Scale bars: 50 µm. n=7 and 6 for wild-type and PTPRT-null mice, respectively. sniffing and direct contact, was analyzed for 3 min. *P<0.05 (Student’s t-test). (G) GABA intensity was decreased in hippocampal CA3 of PTPRT-null mice compared with wild-type mice, but not significantly. Three-chamber sociability test Scale bars: 100 µm. n=7 and 6 for wild-type and PTPRT-null mice, The three-chamber social interaction test box composed of one center respectively. (H) GAT3 expression was reduced significantly in the chamber and two side chambers (each chamber was 30×19 cm) (Sauer hippocampus of PTPRT-null (RT−/−) and D401A-mutant (401MT) mice. et al., 2015). Between each chamber, there was a door (6×6 cm). Stranger Expression is quantified relative to the β-actin control and normalized to the wild-type expression level. n=13 and 15 in wild-type and PTPRT-null mice, boxes consisted of wire cups with an 8.5 cm diameter. The test consisted of respectively; n=11 and 8 in wild-type and D401A-mutant mice, respectively. four steps. First, a mouse was habituated in the chamber for 10 min with *P<0.05, **P<0.01 (Student’s t-test). (I) GAT1 expression was not altered in the the doors open. Next, the mouse was gently placed in the center chamber, hippocampus of PTPRT-null and D401A-mutant mice. Expression is and the doors were closed. A stranger box was then installed in each of the quantified relative to the β-actin control and normalized to the wild-type two side chambers. In the second step, the doors were then opened for expression level. n=7 for wild-type, PTPRT-null mice and D401A-mutant mice. 10 min; this was the social box habituation step. Then, the mouse was (J) In rat synaptosome, GAT3 was pulled down by anti-PTPRT antibody (IP: gently placed in the center chamber, the doors were closed and a same RT), whereas GAT1 was not. Input, 5%. Quantification: GAT3, 54.7; GAT1, gender novel mouse (novel mouse 1) was placed in the novel box on one 18.1; PTPRT, 943.1 (immunoprecipitated protein as a percentage of input side. The doors were then opened for 10 min; this third step was the social protein). (K) When expressed in heterologous cells, GAT3 and PTPRT were interaction step. Finally, the doors were closed and a same gender novel pulled down by anti-PTPRT and anti-GAT3 antibodies, respectively. Input, 3%. mouse was placed in the empty chamber [novel mouse 1 (familiar) versus (L) The interaction of GAT3 with D401A-mutant PTPRT (RT-401) was weaker novel mouse 2 (novel)]. The doors were then opened for 10 min in the last than with wild-type PTPRT (RT-WT) in the GAT3 immunoprecipitation with step of the test, the social novelty step. The social interaction test was lysate from heterologous cells. Input, 3%. Quantification: 0.66, RT-401/RT-WT measured by using a computerized video tracking system, SMART immunoprecipitated by GAT3. (M) Increased tyrosine phosphorylation of GAT3 (Panlab, Barcelona, Spain). in the brain of PTPRT-null mice. After immunoprecipitation with anti- phosphotyrosine antibody, western blotting was performed with anti-GAT3 and Morris water maze test anti-GAT1 antibodies. The tyrosine phosphorylation of GAT3 increased The Morris water maze (MWM) is 122 cm in diameter and is constructed significantly in the brain of PTPRT-null mice (−/−), whereas GAT1 was not. of a stainless steel tank. A Perspex platform (14 cm in diameter and 49 cm 4G10, anti-phosphotyrosine antibody; 4G10+pY, anti-phosphotyrosine antibody pre-incubated with O-phospho-L-tyrosine. The ratio of signal in in height) was submerged 1.5 cm below the surface of the water in PTPRT-null samples to signal in wild-type samples was quantified for the input the middle of a quadrant. White tempera paint was added to the water. and the immunoprecipitation with 4G10 for GAT3 (n=11 and 10 for PTPRT-null The water temperature was maintained at 26°C. The pool was surrounded and wild-type mice, respectively) and GAT1 (n=6 for both PTPRT-null and wild- by curtains, and the mice underwent 8 days of training with four training type mice). **P<0.01 (Student’s t-test). (N) Rescued inhibitory synaptic trials of 90 s (inter-trial interval = 60 s) per day. The platform was transmission of dentate gyrus granule cells by allopregnanolone treatment consistently placed in the same spatial location of the pool throughout (Allo; 30 nM) compared to vehicle treatment (Veh). The frequency of sIPSC of the acquisition period. During each trial, a mouse was released into the PTPRT-null mice increased significantly to the level of wild-type mice by water facing the wall of the pool. After climbing the platform, the mouse addition of allopregnanolone. Allopregnanolone, F(1,50)=5.340, P<0.05; was allowed to stay on it for 30 s. Recording and analysis were performed genotype, F(1,50)=3.607, P=0.63; allopregnanolone × genotype, F(1,50)=0.484, with an Ethovision video tracking system (Noldus Information P=0.490; n=10 and 15 in wild-type and PTPRT-null mice, respectively. Technology, the Netherlands). The escape latency was analyzed by *P<0.05 (two-way ANOVA with Bonferroni post hoc tests). (O) Anti-depression measuring the time taken for the mouse to reach the platform during an effects of allopregnanolone. Intraperitoneal injection of allopregnanolone acquisition trial. (10 mg/kg) effectively relieved PTPRT-null mice from depression-like behavior in the TST. Allopregnanolone, F(1,28)=16.89, P=0.0003; genotype, Chronic restraint stress F(1,28)=46.62, P<0.0001; allopregnanolone × genotype, F(1,28)=0.03, To provide restraint, an 8-week-old mouse weighing 22–23 g was first placed P=0.8726; n=7 and 9 in wild-type and PTPRT-null mice, respectively. *P<0.05, in a ventilated 50 ml conical tube, plugged with a 3-cm-long intermediate tube – – **P<0.01 (two-way ANOVA with Bonferroni post hoc tests). Data in A I, M O and finally placed into a 50 ml tube (Chiba et al., 2012). The mouse could not are mean±s.e.m. move forward or backward on this device. This restraint stress was delivered to the animal for 2 h at the set time every day starting at 10:00 am. A control both objects were calculated. A discrimination index was calculated for each mouse remained in the original cage and remained intact in this home animal and expressed using the following formula, using values from day 2: environment. After binding stress management, the arrested animals were put ðexploring the novel object exploring the familiar object) . back together and returned to their normal home environment. This procedure ðexploring the novel object þ exploring the familiar object) was repeated for 14 days. Journal of Cell Science

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Fig. 8. Attenuated hippocampal neurogenesis in PTPRT-deficient mice. (A) The number of constant dividing nerve cells (BrdU-positive) in the dentate gyrus was not altered in PTPRT-null mice compared with wild-type mice. Scale bar: 100 µm. n=5 for both wild-type and PTPRT-null mice. (B) Newborn granule cells expressing doublecortin (DCX) decreased in PTPRT-null mice. The number and neurite length of DCX-positive cells in the dentate gyrus decreased significantly in PTPRT-null mice compared with wild-type mice. n=12 and 11 in wild-type and PTPRT-null mice, respectively. **P<0.01, ***P<0.001 (Student’s t-test). (C) The number and neurite length of DCX-positive cells in the dentate gyrus decreased significantly in D401A-mutant mice (PTPRT401MT) compared with wild-type mice (PTPRTWT). n=6 for both wild-type and D401A-mutant mice. *P<0.05, ***P<0.001 (Student’s t-test). Scale bar for B and C: 100 µm. (D) Attenuated proliferation of hippocampal NSCs in PTPRT-null mice. The size and the number of neurospheres decreased in PTPRT-null mice compared with wild-type mice. Scale bar: 500 µm. n=3 for both wild-type and PTPRT-null mice. **P<0.01 (Student’s t-test). (E) Aberrant differentiation of neurons from NSCs of PTPRT-null mice. The number of neurons differentiated from hippocampal NSCs (Tuj1-stained, green) was significantly reduced in PTPRT-null mice compared with wild-type mice, but the number of astrocytes differentiated from NSCs (GFAP-stained, red) was not altered. Cells were counterstained with Hoechst 33258 (blue). Scale bar: 100 µm. n=3 for both wild-type and PTPRT-null mice. **P<0.01 (Student’s t-test). (F) Attenuated development of neurons derived from hippocampal NSCs of PTPRT-null mice. The length of dendrites decreased and dendritic arborization was attenuated in neurons derived from hippocampal NSCs of PTPRT-null mice compared with wild-type mice. n=12 for both wild-type and PTPRT-null mice. *P<0.05, **P<0.01 (Student’s t-test). (G) Neurotrophin receptors interacted with PTPRT. BDNF receptor Ntrk2 was pulled down effectively by anti-PTPRT antibody (IP:RT) in rat synaptosomes. p75NTR, PIK3CA, PDPK1 and Akt1 were also pulled down by anti-PTPRT antibody. Input, 5%. Quantification: Ntrk, 134.8; Ntrk2, 623.7; p75NTR, 58.8; PIK3CA, 73.4; PDPK1, 72.3; AKT1, 71.6; PTPRT, 664.2 (immunoprecipitated protein as a percentage of input). (H) Interaction between PTPRT and Ntrk2. When expressed in heterologous cells, PTPRT was pulled down effectively by Ntrk2. Input, 2.5%. Quantification: PTPRT, 52.3 (immunoprecipitated protein as a percentage of input). (I) PTPRT phosphorylated by Ntrk2 tyrosine kinase. Immunoprecipitation with anti-PTPRT antibody followed by western blotting with anti-phosphotyrosine antibody (pY) showed that PTPRT was phosphorylated effectively by Ntrk2. Note that the phosphotyrosine level of wild-type PTPRT (PTPRT-WT) was reduced compared with the activity-dead PTPRT (PTPRT-2CS) because of auto-dephosphorylation. Input, 2.5%. Quantification: PTPRT-WT, 43.0; PTPRT-2CS, 106 (pY signal as a percentage of PTPRT signal). (J) Decreased phosphorylation of Akt1 in the hippocampal tissues of PTPRT-null mice (RT−/−). Phospho-Akt1 (Ser124) decreased significantly in PTPRT-null mice compared with wild-type mice. Ctx, cortex samples; Hippo, hippocampal samples. n=7 and 6 in wild-type and PTPRT-null mice, respectively. *P<0.05 (Student’s t-test). (K) Phospho-PDPK1 (Ser241) decreased significantly in the hippocampal tissues of PTPRT-null mice compared with wild-type mice. n=7 and 6 in wild-type and PTPRT-null mice, respectively. *P<0.05 (Student’s t-test). (L) Phospho-Akt (Ser472) and phospho-S6 (Ser240/244) decreased significantly in the NSCs of PTPRT-null mice compared with wild-type mice. GAPDH was used as a loading control. n=3 for both wild-type and PTPRT-null mice. *P<0.05 (Student’s t-test). Data in A–F, J–L are mean±s.e.m.

Immunoblot analysis Primary neuron culture, transfection, image acquisition and Immunoblotting was performed as previously described (Kim et al., quantification 2016). Briefly, mice were sacrificed and the brain tissue was quickly Primary cultured hippocampal neurons were prepared from P1 mice brains removed and homogenized in a homogenization buffer (50 mM Tris-HCl as described previously (Cheon et al., 2017). Briefly, dissected hippocampi pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS and 0.1% sodium were dissected with trypsin and plated on coverslips coated with poly-L- deoxycholate) containing protease inhibitor cocktail (Roche, Mannheim, lysine in Neurobasal medium (Thermo Fisher Scientific) supplemented with Germany). NSCs were lysed with RIPA buffer, and 30 µg of NSC protein B27 (Thermo Fisher Scientific). After 2–3 h of incubation, the plating lysates was used for each analysis. Protein samples were resolved using medium was changed with a growth medium (plating medium and SDS–PAGE and then transferred onto a polyvinylidene fluoride glutamate). Cultured hippocampal neurons (day in vitro, DIV 7–8) were membrane (Bio-Rad, CA, USA). Blots were incubated with primary and transfected by the calcium phosphate method. For immunofluorescence secondary antibodies followed by visualization using an Enhanced staining, after 7–9 days of transfection, cultured hippocampal neurons were Chemiluminescence kit (Atto Corp., Japan). Immunoblot images were fixed in 4% (v/v) formaldehyde and 4% (w/v) sucrose, and permeabilized quantified using Quantity One 1-D analysis version 4.6.1 software with 0.2% (v/v) Triton X-100 in phosphate-buffered saline (PBS) followed (Bio-Rad Laboratories, Inc., CA, USA) or ImageJ software (NIH, by incubation with primary antibodies and fluorophore-conjugated

Bethesda, MD, USA). secondary antibodies. Images captured by confocal microscopy (LSM Journal of Cell Science

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810, Zeiss) were analyzed blindly using MetaMorph software. The density Immunohistochemistry and image acquisition of dendritic spine and synaptic protein clusters were measured from ∼27–32 Immunohistochemistry was performed as described previously (Go et al., dendrites of ∼7–9 neurons. 2018). Briefly, mice were deeply anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde in PBS. Brains were removed, Cresyl violet staining postfixed overnight, and then cut into 40-μm coronal sections with a The mice were deeply anesthetized and transcardially perfused with saline vibratome (Vibratome VT1200S, Leica Microsystems GmbH, Wetzlar, followed by 4% paraformaldehyde in PBS. Brains were removed, postfixed Germany). Free-floating sections were incubated in PBS containing 3% H2O2 overnight, and then cut into 40-μm coronal sections with a vibratome (v/v), rinsed three times in PBS and blocked with 5% horse serum or goat (Vibratome VT1200S, Leica Microsystems GmbH, Wetzlar, Germany). The serum for 1 h at room temperature. Sections were incubated overnight at 4°C sections were stained with 1% cresyl violet: sections were washed, with primary antibody. After washing, the sections were incubated with dehydrated, cleared, mounted and coverslipped using mounting medium biotinylated secondary anti-rabbit IgG (Vector Laboratories, Inc., (Canada Balsam, Sigma-Aldrich). The slides were dried, after which Burlingame, CA, USA) followed by incubation with avidin-biotinylated photomicrographs were taken using a compound light microscope peroxidase complex (ABC kit, Vector Laboratories, Inc.) and 3,3′- (Olympus, Japan) with 1.25× and 4× objectives (total magnification of diaminobenzidine (Sigma-Aldrich Co. LLC). Immunofluorescence staining 12.5× and 40×). was performed with an Alexa Fluor 488 goat anti-rabbit IgG antibody (secondary antibody; 1:200; Life Technologies Corporation, Grand Island, Mutagenesis of expression plasmids NY, USA). Immunoreactive cells in the hippocampus were counted under a Full-length human PTPRT (NM_133170, amino acids 1–1460) was microscope (Olympus Corporation, Tokyo, Japan) by a blinded observer. subcloned into GW1-CMV and the ectodomain of PTPRT was fused with human Fc domain at the C-terminus, as described previously (Lim et al., Neural stem cell culture 2009). The PTPRT-D401A and PTPRT-D401A-ecto-Fc plasmids Neural stem cells (NSCs) were dissociated from the hippocampus of ∼ – expressing D401A-mutated full-length PTPRT and D401A-mutated embryonic mouse brain (embryonic day 15 16) by trypsinization (Kim human-Fc-fused PTPRT ectodomain were generated using the et al., 2018). Cells were plated into an ultra-low attachment culture dish QuickChange Site-directed Mutagenesis kit (Agilent Technologies, CA, (Corning, Corning, NY) for neurosphere formation and maintained with NSC USA). medium containing DMEM/Neurobasal medium (Thermo Fisher Scientific), 1% N2, 2% B27 (Thermo Fisher Scientific), 20 ng/ml FGF (R&D systems, Viral expression of PTPRT shRNA and PTPRT Minneapolis, MN) and 20 ng/ml EGF (Thermo Fisher Scientific). Lentivirus containing shRNA for knockdown of PTPRT (GGAACCA- TGATAAGAATC), and AAV containing full-length PTPRT were produced NSC proliferation and differentiation assay using GFP-expression shuttle vectors (Lim et al., 2009). To achieve the To measure the proliferation of cultured NSCs, neurospheres were dissociated expression of PTPRT shRNA and PTPRT in dentate gyrus, we bilaterally into single cells by trypsinization, and dissociated cells (1000 cells/well) were – microinjected 1 μl of lentivirus or AAV at the dentate gyrus of three-week- plated into ultra-low attachment 24-well plates. After 6 10 days, secondary old wild-type and knockout mice (−1.8 mm AP, ±1.5 mm ML, −2.0 mm neurospheres were trypsinized, and the dissociated cell number was counted. DV). GFP-expressing neurons were shown in the dentate gyrus area To measure the multi-lineage differentiation ability of NSCs, cultured (Fig. 2F,I). After five weeks, behavioral experiments were performed. neurospheres were dissociated by trypsinization and transferred into 24-well plates coated with 50 µg/ml poly-D-lysine (Sigma, St. Louis, MO) and 10 µg/ ml laminin (Sigma). NSC differentiation was induced by incubating the cells Stereotaxic surgery with differentiation medium containing DMEM/F12 with N2 and B27 Three-week-old male mice were anaesthetized with intraperitoneal – injections of ketamine and xylazine (61.8 mg/kg and 6.13 mg/kg, without FGF or EGF. After 5 6 days, cells were fixed with 4% respectively) and mounted on stereotaxic apparatus (Stoelting Europe, paraformaldehyde solution. Differentiated neurons and astrocytes were Dublin, Ireland). Injections were made with a 10 µl syringe, connected to the visualized by immunofluorescence staining with anti-Tuj1 (Biolegend, San injector by a polyethylene tubing, and controlled by an injection pump at Diego, CA) and anti-GFAP (Thermo Fisher Scientific, Waltham, MA) 0.5 µl/min. antibodies, respectively. The length of neuronal axons and dendrites and neuronal arborization were measured using ImageJ software. Tuj1- and GFAP-positive cells were also counted using ImageJ software. Analysis of AMPA receptor surface trafficking Cultured hippocampal neurons transfected with HA-tagged GluR1 and GluR2 (generously provided by Dr Sheng, Broad Institute of MIT and Preparation of hippocampal slices for patch clamp Harvard, USA) and with PTPRT-shRNA-pSup.gfp (Lim et al., 2009) were Mice were decapitated under deep enflurane anesthesia, and the brains were fixed with 4% (v/v) formaldehyde and 4% (w/v) sucrose for 8 min at room quickly removed and transferred to ice-cold dissection buffer containing temperature, and incubated with mouse anti-HA antibody at 4°C overnight. sucrose (212.7 mM), KCl (2.6 mM), NaH2PO4 (1.23 mM), NaHCO3 The next day, neurons were washed and incubated with Cy3-conjugated (26 mM), dextrose (10 mM), MgCl2 (10 mM) and CaCl2 (0.5 mM). Coronal anti-mouse secondary antibody. Then neurons were fixed with ice-cold whole brain sections (300 µm in thickness) prepared using a vibratome methanol at −20°C for 1 m 30 s, and washed, followed by incubation with (Vibratome 1000 plus, The Vibratome Company, St Louis, MO, USA) were rabbit anti-HA antibody and guinea pig anti-GFP antibody at room placed into dissection buffer that was continuously bubbled with 5% CO2/95% temperature for 3 h. Neurons were then washed and incubated with Cy5- O2 (v/v). The hippocampal slices were mainly used for patch clamp. The slices conjugated anti-rabbit and FITC-conjugated anti-guinea pig secondary were held at 35°C for 1 h in a chamber filled with continuously oxygenated antibodies. Images captured by confocal microscopy (LSM 810, Zeiss) were artificial cerebrospinal fluid (ACSF) with the following composition: NaCl analyzed blindly using MetaMorph software. (124 mM), KCl (5 mM), NaH2PO4 (1.25 mM), NaHCO3 (26 mM), dextrose (10 mM), MgCl2 (1.5 mM) and CaCl2 (2.5 mM). The slices were then ˚ BrdU incorporation transferred to an open submersion-type recording chamber, maintained at 35 C, 5-bromo-2-deoxyuridine (BrdU; 150 mg/kg/day) was intraperitoneally injected and perfused with oxygenated ACSF at a flow rate of 2 ml/min. for three consecutive days. After 2 days, mice were deeply anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde in PBS. Induction of long-term potentiation and long-term depression, Brains were removed, postfixed overnight, and then cut into 40-μm coronal and measurement of paired-pulse facilitation sections with a vibratome (Vibratome VT1200S, Leica Microsystems GmbH, A bipolar stimulating electrode was inserted into Schaffer Collaterals or Wetzlar, Germany). An immunohistochemical assay was performed with a perforant path to activate CA1 pyramidal cells or dentate gyrus granule cells primary anti-BrdU antibody (AbD Serotec, CA, USA). in the hippocampus, respectively. A glass micropipette filled with ACSF Journal of Cell Science

16 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243972. doi:10.1242/jcs.243972 was inserted into CA1 stratum radiatum or the granule cell layer of dentate The supernatant was obtained by centrifugation, diluted by tenfold using gyrus to record field potentials (FPs). FPs were evoked by stimulating the 0.02 N HCl, mixed with 5% (v/v) trichloroacetic acid at ratio of 1:4 in order to Schaffer Collaterals with an electrical pulse 0.2 ms in duration, delivered obtain free amino acids. This supernatant was filtered and injected into an ion- through concentric bipolar stimulating electrodes (FHC, Bowdoinham, exchange column. Glutamate (Glu) and GABA were separated after 10 min. ME). The initial slope of extracellular FPs was recorded in the CA1 stratum The resulting chromatogram identified each monoamine position and radiatum. Baseline responses were obtained upon application of 50% of the concentration from the sample as compared to that of the standard, and maximal stimulation at 0.033 Hz. Long-term potentiation (LTP) of CA1 was finally, the calculation of the content of each monoamine as µg/l in brain tissue induced using a conventional stimulation paradigm; the theta burst was made according to established procedures (Pagel et al., 2000). stimulation (TBS) protocol consisted of eight bursts, each of four 100 Hz Physiological solution determination could be carried out fully automatically pulses, administered at 200 ms intervals. For LTP of dentate gyrus, four in just 110 min. TBSs were applied in the presence of bicuculline. The stimulus intensity during TBS was identical to that of the test pulse. All measurements were Analysis of phosphotyrosine within brain synaptosome expressed as percentages of the average values calculated 20 min prior to Anti-phosphotyrosine antibody (4G10) agarose conjugate (Millipore, LTP induction. Significant differences between groups were sought via Germany) was used for immunoprecipitation to analyze the level of evaluation of average LTP values 58–60 min after LTP. Low-frequency tyrosine phosphorylation of synaptic molecules. 4G10 agarose conjugate stimulation consisting of 900 pulses at 1 Hz for 15 min was applied to was incubated with synaptosome at 4°C for 4 h, and washed followed by induce long-term depression (LTD). To measure paired-pulse facilitation boiling with SDS sample buffer. Protein samples were resolved using (PPF), we used inter-stimulus intervals (ISIs) of 25 ms, 50 ms, 100 ms, SDS–PAGE and immunoblotted with primary antibodies. 200 ms, 400 ms, 1000 ms and 2000 ms. Interaction between the ectodomains of PTPRT Recording of spontaneous- and miniature-excitatory PTPRT expressed in heterologous cells was recruited by the Fc-fused postsynaptic currents, and spontaneous- and miniature- PTPRT-ecto (RT-Fc) as described previously (Lim et al., 2009). Briefly, for inhibitory postsynaptic currents, in the ventral hippocampal trans interactions, purified RT-Fc pre-bound on protein A-beads was used dentate granule cells for pulldown of PTPRT expressed in heterologous cells. For trans+cis The ventral hippocampal slices were perfused with ACSF at a ∼2 ml/min flow. interactions, purified RT-Fc was mixed with PTPRT expressed in Data was recorded at a holding potential of −70 mV using an axopatch 700B heterologous cells, and then protein A-beads were added for pulldown. amplifier (Axon Instruments, Foster City, CA, USA), and ACSF temperature For cis interactions, RT-Fc was co-expressed with PTPRT in heterologous was maintained at 35°C. Current output of excitatory postsynaptic currents cells, and then a pulldown assay was performed with protein A-beads. (EPSCs) was filtered at 1 kHz and digitized at 10 kHz. For recording of spontaneous EPSCs (s-EPSCs), the recording pipette was filled with internal Statistical analysis solution containing 120 mM CsMeSO3,5mMMgCl2,8mMNaCl,1mM GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) EGTA, 10 mM HEPES, 0.001 mM QX-314, 0.5 mM Na3 GTP and 2 mM Mg was used to perform all statistical analyses. Two-sample comparisons ATP (pH 7.2–7.3, 280–290 mOsm), and 20 µM 1(s),9(R)-(−)-Bicuculline were conducted with Student’s t-tests, while multiple comparisons methiodide (Bicuculline; Sigma-Aldrich, St. Louis, MO, USA) was added to were performed with a one-way analysis of variance (ANOVA) followed ACSF. To record the miniature EPSCs (mEPSCs), we added additional 1 µM by Tukey post hoc test or a two-way ANOVA followed by Bonferroni tetrodotoxin (TTX; Tocris Bioscience, Ellisville, MO, USA) to ACSF. Current post hoc tests. All results are presented as the mean±s.e.m. output of inhibitory postsynaptic currents (IPSCs) was filtered at 4 kHz and Differences with a P value less than 0.05 were considered to be digitized at 10 kHz. For recording of spontaneous IPSCs (sIPSCs), the statistically significant. recording pipette was filled with internal solution containing 140 mM KCl, 0.5 mM CaCl2,20mMHEPES,5mMEGTAand5mMMgATP(pH7.2– Antibodies 7.3, 310–315 mOsm), and 50 µM DL-2-Amino-5-phosphonopentanoic acid The primary antibodies used were mouse anti-PTPRT (Abfrontier, Cat# LF- (AP5; Sigma-Aldrich, St. Louis, MO, USA) and 20 µM 6-cyano- MA0304; 1:2000), mouse anti-PSD-95 (Thermo Fisher Scientific, Cat# 7nitroquinoxaline-2,3-dione (CNQX; Tocris Bioscience, Ellisville, MO, MA1-045; 1:200), rabbit anti-vGlut1 (SYSY, Cat# MA1-045; 1:500), mouse USA) were added to ACSF. To record the miniature IPSCs (mIPSCs), we anti-gephyrin (SYSY, Cat# 147 111; 1:250), rabbit anti-vGAT (SYSY, Cat# added additional 1 µM TTX to ACSF. All experiments were executed in 3– 131 002; 1:250), rabbit anti-GAT1 (Abcam, Cat# ab426; 1:1000), rabbit anti- 5MΩ resistance of patch electrodes and access resistance <25 MΩ.Datawere GAT3 (Abcam, Cat# ab181783; 1:2000), rabbit anti-Ntrk (Abcam, Cat# acquired using Clampex software (Axon instruments, Union City, CA) at gain ab181560; 1:2000), rabbit anti-Ntrk1 (Cell Signaling Technology, Cat#2505; of 1 for EPSCs and IPSCs. 1:500), rabbit anti-Ntrk2 (Abcam, Cat# ab18987; 1:1000), rabbit anti-Ntrk2 (ecto) (Abcam, Cat#ab33655; 1:1000), rabbit anti-p75NTR (Abcam, Cat# Measurement of neurotransmitters within brain tissues ab52987; 1:2000), rabbit anti-PIK3CA (Cell Signaling Technology, Cat# All of the animals were deeply anesthetized with isoflurane or sodium #4249; 1:1000), rabbit anti-PDPK1 (Cell Signaling Technology, Cat# 3062; pentobarbital (80 mg/kg, intraperitoneal). The brain was rapidly removed from 1:1000), rabbit anti-phospho-PDPK1 (Ser241) (Cell Signaling Technology, the cranium, and brain areas were dissected out on an ice-cold plate. The Cat# 3438; 1:1000), rabbit anti-Akt (Cell Signaling Technology, Cat# 9272; hippocampus was separated, weighed and placed in 1.5-ml microcentrifuge 1:2000), rabbit anti-phospho-Akt1 (Ser124) (Abcam, Cat# ab183556; tubes. For glutamine, glutamate and GABA, the samples from both 1:1000), rabbit anti-phospho-Akt1 (Ser473) (Cell Signaling Technology, hemispheres were removed and pooled in 500 µl of deionized water. The Cat# 4060; 1:1000); rabbit anti-GAPDH (Cell Signaling Technology, Cat# samples were homogenized for 20 s with a sonic dismembrator (Thermo Fisher 2118; 1:1000), mouse anti-S6 ribosomal protein (Cell Signaling Technology, Scientific, USA) and centrifuged (Micromax RF/IEC, Thermo Fisher Cat# 2317; 1:1000), rabbit anti-phospho-S6 ribosomal protein (Ser240/244) Scientific, USA) at 6000 g at 3°C for 20 min. Supernatants were filtered (Cell Signaling Technology, Cat# 5364; 1:1000), goat anti-doublecortin through 0.22 µm hydrophilic filters (Shleicher & Schuell, Germany), and 10 µl (Santa Cruz Biotechnology, Cat# sc-8666; 1:1000), rat anti-5-bromo-2- of brain filtrate was diluted in 990 µl of deionized water for amino acid analysis. deoxyuridine (BrdU) (AbD Serotec, Cat# MCA2060GA; 1:1000), rabbit anti- Finally, samples were stored at approximately −20°C until analysis. An amino calretinin (Millipore, Cat# ab22683; 1:2000), mouse anti-calreticulin acid analyzer (AAA; HITACHI L-8900, Japan) used with autosampling (Abcam, Cat# ab22683; 1:200), rabbit anti-calbindin (Abcam, Cat# injector 20 µl loop and an ultraviolet (UV) variable wavelength detector was ab11426; 1:3000), mouse anti-parvalbumin (Millipore, Cat# MAB1572; used for monoamine assays where the samples were injected directly into a 1:2000), rabbit anti-somatostatin (Thermo Fisher Scientific, Cat# PA5-16253; HITACHI HPLC Packed Post-column (#2622PF; 4.6×60 mm) packed with 1:200), rabbit anti-neuropeptide Y (NPY) (Abcam, Cat# ab30914; 1:200), ion-exchange resin, flow rate 0.5 ml/min, UV 570 nm. The AAA utilizes a rabbit anti-cholecystokinin (CCK) (Sigma-Aldrich, Cat# SAB1402711; lithium citrate buffer system and analyses using the ninhydrin reaction method. 1:200), mouse anti-Tuj1 (Biolegend, Cat# 801201; 1:1000), rabbit anti- Journal of Cell Science

17 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243972. doi:10.1242/jcs.243972

GluR1 and anti-GluR2 (generously provided by Professor Eujoon Kim, Berton, O. and Nestler, E. J. (2006). New approaches to antidepressant drug KAIST, Korea; 1:2000), mouse anti-HA (Roche, Cat# 11 583 816 001; discovery: Beyond monoamines. Nat. Rev. Neurosci. 7, 137-151. doi:10.1038/ 1:200), rabbit anti-HA (Santa Cruz Biotechnology, Cat# sc-805; 1:200) and nrn1846 Besco, J. A., van Huijsduijnen, R. H., Frostholm, A. and Rotter, A. (2006). rat anti-GFAP (Thermo Fisher Scientific, Cat# 13-0300; 1:200). The Cy3-, Intracellular substrates of brain-enriched receptor protein tyrosine phosphatase Cy5-, FITC- and HRP-conjugated secondary antibodies were obtained from rho (RPTPρ/PTPRT). Brain Res. 1116, 50-57. doi:10.1016/j.brainres.2006.07. Jackson ImmunoResearch. Biotinylated secondary anti-rabbit IgG were from 122 Vector Laboratories, Inc. Boldrini, M., Underwood, M. D., Hen, R., Rosoklija, G. B., Dwork, A. J., Mann, J. J. and Arango, V. (2009). Antidepressants increase neural progenitor cells in the human hippocampus. Neuropsychopharm 34, 2376-2389. doi:10.1038/npp. Drugs 2009.75 Imipramine hydrochloride (20 mg/kg, intraperitoneal) was purchased from Boldrini, M., Santiago, A. N., Hen, R., Dwork, A. J., Rosoklija, G. B., Tamir, H., Sigma-Aldrich Co. LLC (St Louis, MO, USA) and diluted in saline. Arango, V. and Mann, J. J. (2013). Hippocampal granule neuron number and Allopregnanolone (3α,5α-tetrahydroprogesterone, 3α,5α-THP; 10 mg/kg, dentate gyrus volume in antidepressant-treated and untreated major depression. intraperitoneal) was purchased from Calbiochem (Cat# 127100), dissolved Neuropsychopharmacology 38, 1068-1077. doi:10.1038/npp.2013.5 in ethanol and diluted in saline. Bond, A. M., Ming, G.-L. and Song, H. (2015). Adult mammalian neural stem cells and neurogenesis: Five decades later. Cell Stem Cell 17, 385-395. doi:10.1016/j. stem.2015.09.003 Methods details Bremner, J. D., Narayan, M., Anderson, E. R., Staib, L. H., Miller, H. L. and For all experiments, mice, cultured neurons and NSCs were randomly Charney, D. S. (2000). Hippocampal volume reduction in major depression. allocated to experimental groups, and all data collected throughout these Am. J. Psychiatry 157, 115-118. doi:10.1176/ajp.157.1.115 studies were included in the analysis. No data were excluded. No sample- Caspi, A., Sugden, K., Moffitt, T. E., Taylor, A., Craig, I. W., Harrington, H. L., size estimates were conducted due to technical limitations on sample McClay, J., Mill, J., Martin, J., Braithwaite, A. et al. (2003). Influence of life collection. All attempts were made to use maximal sample size in each stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, 386-389. doi:10.1126/science.1083968 experiment whenever possible. Chang, C.-Y., Chen, Y.-W., Wang, T.-W. and Lai, W.-S. (2016). Akting up in the GABA hypothesis of schizophrenia: Akt1 deficiency modulates GABAergic Competing interests functions and hippocampus-dependent functions. Sci. Rep. 6, 33095. doi:10. The authors declare no competing or financial interests. 1038/srep33095 Chaudhury, D., Liu, H. and Han, M.-H. (2015). Neuronal correlates of depression. Author contributions Cell. Mol. Life Sci. 72, 4825-4848. doi:10.1007/s00018-015-2044-6 Conceptualization: K.-S.K., S.S.M., J.-R.L.; Methodology: M.-H.K., D.Y.L., J.M., I.S., Cheon, C. K., Lim, S.-H., Kim, Y.-M., Kim, D., Lee, N.-Y., Yoon, T.-S., Kim, N.-S., S.S.M.; Validation: S.-H.L., S.S., K.-S.K.; Formal analysis: S.-H.L., S.S., E.C.K., Kim, E. and Lee, J.-R. (2017). Autosomal dominant transmission of complicated D.Y.L., J.M., H.-Y.P., Y.-K.R., Y.M.K., Y.J.K., T.H.K., N.-Y.L., I.S.; Investigation: hereditary spastic paraplegia due to a dominant negative mutation of KIF1A, SPF30 gene. Sci. Rep. 7, 12527. doi:10.1038/s41598-017-12999-9 K.-S.K., S.S.M., J.-R.L.; Resources: N.-S.K., D.Y.Y., Y.G., M.S.; Data curation: Chiba, S., Numakawa, T., Ninomiya, M., Richards, M. C., Wakabayashi, C. and M.-H.K., E.K., K.-S.K.; Writing - original draft: K.-S.K., S.S.M.; Writing - review & Kunugi, H. (2012). Chronic restraint stress causes anxiety- and depression-like editing: J.-R.L.; Supervision: J.-R.L.; Project administration: J.-R.L.; Funding behaviors, downregulates glucocorticoid receptor expression, and attenuates acquisition: S.S.M., J.-R.L. glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 39, 112-119. doi:10.1016/ Funding j.pnpbp.2012.05.018 This study was supported by grants from the Brain Research Program (NRF- Conrad, C. D., Galea, L. A. M., Kuroda, Y. and McEwen, B. S. (1996). Chronic 2015M3C7A1029113), the Postgenomic Research Program (NRF- stress impairs rat spatial memory on the Y maze, and this effect is blocked by 2014M3C9A2064619), the Bio & Medical Technology Development Program (NRF- tianeptine treatment. Behav. Neurosci. 110, 1321-1334. doi:10.1037/0735-7044. 2016M3A9B6904244) and the Basic Science Research Program (NRF- 110.6.1321 2019R1I1A2A01063642) through the National Research Foundation of Korea Czéh, B., Vardya, I., Varga, Z., Febbraro, F., Csabai, D., Martis, L.-S., Højgaard, (NRF) funded by the Ministry of Science and ICT, South Korea, and by the Ministry of K., Henningsen, K., Bouzinova, E. V., Miseta, A. et al. (2018). Long-term stress Education, the Korea Research Institute of Bioscience and Biotechnology KRIBB disrupts the structural and functional integrity of GABAergic neuronal networks in Initiative Research Program (KGM5222012), and the Ministry of Education, Culture, the medial prefrontal cortex of rats. Front. Cell Neurosci. 12, 148. doi:10.3389/ Sports, Science and Technology KAKEN program (KAKENHI No.17H00789). fncel.2018.00148 David, D. J., Samuels, B. A., Rainer, Q., Wang, J.-W., Marsteller, D., Mendez, I., Drew, M., Craig, D. A., Guiard, B. P., Guilloux, J.-P. et al. (2009). Neurogenesis- Supplementary information dependent and –independent effects of fluoxetine in an animal model of anxiety/ Supplementary information available online at depression. Neuron 62, 479-493. doi:10.1016/j.neuron.2009.04.017 https://jcs.biologists.org/lookup/doi/10.1242/jcs.243972.supplemental Drew, L. J., Kheirbek, M. A., Luna, V. M., Denny, C. A., Cloidt, M. A., Wu, M. V., Jain, S., Scharfman, H. E. and Hen, R. (2016). Activation of local inhibitory Peer review history circuits in the dentate gyrus by adult-born neurons. Hippocampus 26, 763-778. The peer review history is available online at doi:10.1002/hipo.22557 https://jcs.biologists.org/lookup/doi/10.1242/jcs.243972.reviewer-comments.pdf Duman, R. 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