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Neuroligin 3 Regulates Dendritic Outgrowth by Modulating Akt/mTOR Signaling

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Neuroligin 3 Regulates Dendritic Outgrowth by Modulating Akt/mTOR Signaling

Jing Xu et al. Front Cell Neurosci.

Abstract

Neuroligins (NLs) are a group of postsynaptic cell adhesion molecules that function in synaptogenesis and synaptic transmission. Genetic defects in neuroligin 3 (NL3), a member of the NL protein family, are associated with autism. Studies in rodents have revealed that mutations of NL3 gene lead to increased growth and complexity in dendrites in the central nervous system. However, the detailed mechanism is still unclear. In our study, we found that deficiency of NL3 led to morphological changes of the pyramidal neurons in layer II/III somatosensory cortex in mice, including enlarged somata, elongated dendritic length, and increased dendritic complexity. Knockdown of NL3 in cultured rat neurons upregulated Akt/mTOR signaling, resulting in both increased protein synthesis and dendritic growth. Treating neurons with either rapamycin to inhibit the mTOR or LY294002 to inhibit the PI3K/Akt activity rescued the morphological abnormalities resulting from either NL3 knockdown or knockout (KO). In addition, we found that the hyperactivated Akt/mTOR signaling associated with NL3 defects was mediated by a reduction in phosphatase and tensin (PTEN) expression, and that MAGI-2, a scaffold protein, interacted with both NL3 and PTEN and could be a linker between NL3 and Akt/mTOR signaling pathway. In conclusion, our results suggest that NL3 regulates neuronal morphology, especially dendritic outgrowth, by modulating the PTEN/Akt/mTOR signaling pathway, probably via MAGI-2. Thereby, this study provides a new link between NL3 and neuronal morphology.

Keywords: Akt/mTOR; MAGI-2; PTEN; dendritic outgrowth; neuroligin 3.

Figures

FIGURE 1
FIGURE 1
Changes in dendritic outgrowth in NL3 KO somatosensory cortex. (A) Golgi staining of somatosensory cortex in NL3 KO (right) and WT (left) littermates at 3 months of age. Layer V and VI were indicated. Scale bar, 50 μm. (B) Representative tracings of Golgi-stained pyramidal neurons in layer II/III somatosensory cortex. Left, wild type; right, NL3 KO. Scale bar, 50 μm. Quantifications of the soma area (C), total dendritic length (D), and sholl-analysis for dendritic arborization (E) of the pyramidal neurons from layer V/VI somatosensory cortex. Values represent mean ± SEM (for C, n = 147 and 170 neurons, respectively, and for D and E, n = 25 and 18 pyramidal neurons, respectively, from three pairs of brains; ∗∗∗p < 0.001, ∗∗p < 0.01, unpaired two-tailed Student’s t-test).
FIGURE 2
FIGURE 2
Overactivation of mTOR signaling and abnormal dendritic outgrowth in neurons with NL3 knockdown. (A) Western blots of cultured rat hippocampal neurons infected by shNL3-expressing lentivirus or the control lentivirus at DIV5, and harvested at DIV10. Ctrl, control; shNL3, NL3-shRNA. (B) Quantification of the NL3 expression as in A. NL3 expression of shNL3 infected neurons was reduced by 76% compared with control. Values represent mean ± SEM normalized to control (n = 6 independent experiments; ∗∗∗p < 0.001, one-sample t-test). (C) Immunostaining showed that the NL3 expression was enormously reduced in neurons infected by shNL3 lentivirus. Cultured rat hippocampal neurons infected with related lentivirus at DIV5 were immunostained at DIV10 using NL3-specific antibodies. Scale bar, 20 μm. (D) NL3 knockdown leads to hyperphosphorylation of S6. Immunoblots of cultured rat hippocampal neurons infected by shNL3-expressing lentivirus or the control lentivirus at DIV5, and harvested at DIV10. Ctrl, control; shNL3, NL3-shRNA. (E) Quantifications of the levels of NL3 and S6 phosphorylation as in D. The phosphorylated S6 protein level of the NL3 knockdown group was higher than that of the control group. Values represent mean ± SEM, normalized to control (n = 5 independent experiments; ∗∗∗p < 0.001, p < 0.05, one-sample t-test). (F) NL3 knockdown promotes protein synthesis in neurons. Immunoblots of cultured rat hippocampal neurons infected with shNL3-expressing or control lentivirus at DIV5, incubated with puromycin at DIV10, and examined for newly synthesized proteins using anti-puromycin antibody. (G) Quantification of blots as in F. The expression level of puromycin-labeled proteins from neurons with NL3 knockdown was higher than that from control neurons. Values represent mean ± SEM, normalized to control (n = 3 independent experiments; p < 0.05, one-sample t-test). (H) NL3 knockdown affects the outgrowth of dendrites. Photomicrographs of cultured rat hippocampal neurons at DIV6 which were infected with lentivirus co-expressing GFP and shNL3, or vector control at DIV1. GFP signal indicates successful viral infection. Green, GFP; red, MAP2. Scale bar, 20 μm. (I) Quantifications of soma size, total dendritic length, and numbers of dendritic terminals and primary dendrites based on MAP2 signals. The neurons in the NL3 knockdown group showed significantly longer dendrites. Values represent mean ± SEM (n = 3 independent experiments; ∗∗p < 0.01, paired two-tailed Student’s t-test).
FIGURE 3
FIGURE 3
Upregulation of Akt activity in neurons with NL3 knockdown. (A) NL3 knockdown increased Akt phosphorylation level. Cultured rat hippocampal neurons were infected by shNL3 or control lentivirus at DIV5, and harvested for western blot analysis at DIV10. p-Akt-473, phosphorylation of Akt Ser473 residue; p-Akt-308, phosphorylation of Akt Thr308 residue. (B) Quantifications of the levels of NL3 and Akt Thr308/Ser473 phosphorylation as in A. The phosphorylation levels of both Thr308 site and Ser473 site of Akt were significantly increased in neurons with NL3 knockdown. Values represent mean ± SEM, normalized to control (n = 5 independent experiments; ∗∗p < 0.01, p < 0.05, one-sample t-test). (C,E) NL3 knockdown increased the phosphorylation levels of both Thr308 and Ser473 sites of Akt. Cultured rat hippocampal neurons infected with related lentivirus at DIV5 were immunostained at DIV10 using phospho-specific antibodies as indicated. Scale bar, 20 μm. Quantifications of the intensities of phosphorylated Akt Ser473 (D) and Thr308 (F) as in C and E, respectively. The signal intensities of both phosphorylated Akt Ser473 (D) and Thr308 (F) were higher in the NL3 knockdown neurons than in their controls. Values represent mean ± SEM normalized to control (n = 3 independent experiments; ∗∗p < 0.01, paired two-tailed Student’s t-test).
FIGURE 4
FIGURE 4
Upregulation of Akt activity and morphological changes in cultured NL3 knockout cortical neurons. (A) Western blot showed that Akt phosphorylation at Thr308 was increased in NL3 knockout cortical neurons. The neurons were cultured from NL3 KO newborn mice or WT littermates at P0 and harvested to be immunoblotted at DIV6. WT, wild type; KO, NL3 knockout. (B) Quantifications of blots as in A. The phosphorylation level of Akt at Thr308 sites was significantly increased in NL3 knockout neurons. Values represent mean ± SEM normalized to control (n = 5 independent experiments; ∗∗p < 0.01, p < 0.05, one-sample t-test). (C,D) NL3 knockout cortical neurons were processed for immunocytochemistry assay at DIV6 using anti-MAP2 antibody and anti-phospho-specific antibody of Ser473 or Thr308. Scale bar, 20 μm. (E) Quantifications of the intensity of phosphorylated Akt at Ser473 site as in C. NL3 knockout neurons showed similar intensity of the phosphorylated Akt at Ser473 site with WT neurons. Values represent mean ± SEM normalized to control (n = 3 independent experiments; p < 0.05, paired two-tailed Student’s t-test). (F) Quantifications of the intensity of phosphorylated Akt at Thr308 as in D. The intensity of the phosphorylated Akt at Thr308 was higher in NL3 knockout neurons than in WT neurons. Values represent mean ± SEM normalized to control (n = 3 independent experiments; p < 0.05, paired two-tailed Student’s t-test). (G) Abnormal morphological changes in NL3 KO cortical neurons. Photomicrographs of cortical neurons cultured from NL3 knockout mice were processed for immunostaining using MAP2 antibody to label dendrites at DIV6. Scale bar, 20 μm. (H) Quantifications of soma size, total dendritic length, and numbers of dendritic terminals and primary dendrites. Compared with WT neurons, NL3 KO neurons showed larger soma size, longer dendritic length, and increased number of dendritic tips. Values represent mean ± SEM (n = 4 independent experiments; ∗∗p < 0.01, p < 0.05, paired two-tailed Student’s t-test).
FIGURE 5
FIGURE 5
Pharmacological inhibition of mTOR signaling rescued the morphological abnormalities of neurons with NL3 deficiency. (A) NL3 knockdown-induced hyperactivation of Akt/mTOR signaling was inhibited by rapamycin and LY294002. Cultured rat hippocampal neurons were infected by shNL3 or control lentivirus at DIV1, incubated with DMSO, rapamycin (10 nM), or LY294002 (10 μM) for 24 h at DIV5, and then harvested and processed for western blot analysis using phospho-specific antibodies of Akt, mTOR, and S6. rapa, rapamycin; LY, LY294002. (B) Quantifications of the phosphorylation levels of the proteins involved in Akt/mTOR signaling pathway as in A. Values represent mean ± SEM, normalized to control (n = 5 independent experiments; ∗∗p < 0.05, p < 0.05, one-sample t-test; ###p < 0.001, ##p < 0.01, #p < 0.05, two-way ANOVA with post hoc Bonferroni test). (C) Rapamycin and LY294002 rescued the neuronal morphological abnormalities induced by NL3 knockdown. Neurons with lentiviral infection at DIV1 were treated with rapamycin (10 nM), or LY294002 (10 μM) for 24 h at DIV5, and then processed for immunostaining using anti-MAP2 antibody to label dendrites. Scale bar, 20 μm. (D) Quantifications of the total dendritic length of the cultured neurons as in C. The neuronal dendrites of the NL3 knockdown group were longer than that of the control group. This increase was eliminated after either rapamycin or LY294002 treatment. Values represent mean ± SEM (n = 3 independent experiments; ∗∗p < 0.01, one-way ANOVA with Tukey’s post hoc test). (E) The abnormal structural changes in NL3 KO cortical neurons were reversed by rapamycin or LY294002. Cortical neurons cultured from NL3 KO mice were incubated with rapamycin (10 nM), or LY294002 (10 μM) for 24 h at DIV5, and processed for immunostaining using anti-MAP2 antibody to label dendrites. Scale bar, 20 μm. (F) Quantifications of the soma size, the total dendritic length, and the number of dendritic tips of the neurons as in E. The enlarged soma size, elongated dendritic length, and increased number of dendritic tips in NL3 KO neurons were eliminated by rapamycin treatment for NL3 knockout neurons, while LY294002 treatment rescued the enlarged soma size and elongated dendritic length in the knockout neurons. Values represent mean ± SEM (n = 4 independent experiments; ∗∗p < 0.01, p < 0.05, one-way ANOVA with Tukey’s post hoc test).
FIGURE 6
FIGURE 6
PTEN expression level is down-regulated by NL3 knockdown. (A) Western blot showed that NL3 knockdown reduced the protein level of PTEN. Cultured rat hippocampal neurons were infected by shNL3 or control lentivirus at DIV5, and harvested and immunoblotted at DIV10 using antibody against PTEN as indicated. (B) Quantification of blots as in A. The expression level of PTEN was significantly reduced after NL3 knockdown. Values represent mean ± SEM normalized to control (n = 5 independent experiments; ∗∗p < 0.01, one-sample t-test). (C) Immunostaining showed that NL3 knockdown resulted in reduced PTEN expression. Cultured rat hippocampal neurons were infected by shNL3 or control lentivirus at DIV1, and immunoreactived to PTEN (red) and to MAP2 (blue) at DIV6. Scale bar, 20 μm. (D) Quantification of PTEN expression level as in C. The PTEN expression of the NL3 knockdown group was significantly lower than control. Values represent mean ± SEM normalized to control (n = 3 independent experiments; ∗∗p < 0.01, paired two-tailed Student’s t-test).
FIGURE 7
FIGURE 7
NL3 and PTEN formed a complex via MAGI-2. (A,B) Co-immunoprecipitation assays confirmed the interaction between NL3 and MAGI-2. HEK293T cells co-transfected with HA-NL3 and Myc-MAGI-2 plasmids or control plasmids were processed for co-IP assays using either NL3 antibody or myc antibody. Myc-MAGI-2 was pulled down together with HA-NL3 (A), so was HA-NL3 by Myc-MAGI-2 (B). (C) Myc-MAGI-2 was co-localized with HA-NL3. Photomicrographs showed that HA antibody immunostained HA-NL3 (green) is localized specifically at plasma membrane, while myc antibody immunostained myc-MAGI-2 (red) was diffusely distributed in cytoplasm in HEK293T. When myc-MAGI-2 was co-transfected with HA-NL3, it was transported to plasma membrane and co-localized with HA-NL3. Scale bar, 10 μm. (D) Co-immunoprecipitation assays verified the interaction between MAGI-2 and PTEN. HEK293T cells co-transfected with myc-MAGI-2 and GFP-PTEN plasmids or control plasmids were processed for co-IP assays using myc antibody. GFP-PTEN was pulled down together with myc-MAGI-2. (E) Co-immunoprecipitation assays in HEK293T detected that NL3 and PTEN formed a complex via MAGI-2. HEK293T cells co-transfected with NL3, GFP-PTEN, and myc-MAGI-2 or control plasmids were processed for co-IP assays using GFP antibody. NL3 and myc-MAGI-2 were pulled down together with GFP-PTEN.
FIGURE 8
FIGURE 8
The schematic model of the process of regulating Akt/mTOR signaling by NL3. NL3 recruiting MAGI-2 to plasma membrane which could bind and stabilize PTEN to suppress the Thr308 phosphorylation of Akt catalyzed by PI3K. Akt activation is required not only the phosphorylation of Thr308 site, but also the phosphorylation of Ser473 site by mTORC2. Activated Akt is able to activate mTORC1 directly or via TSC/Rheb pathway. The activated mTORC1 phosphorylates its downstream kinase S6K1 which regulates protein synthesis by activating the substrate S6.

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