Studies have shown that peroxisome proliferator-activated receptor γ (PPAR γ) is down-regulated in pulmonary vascular lesions of patients with pulmonary hypertension (PH) and animal models of PH. Yet, the detailed molecular mechanisms underlying this alteration are not fully defined; the aim of this study is to address this issue. 5-HT dose- and time-dependently reduced PPAR γ expression and promoted pulmonary artery smooth muscle cells (PASMCs) proliferation; this was accompanied with the phosphorylation of Akt, inactivation of GSK-3β and up-regulation of β-catenin. Importantly, pre-treatment of cells with PI3K inhibitor (Ly294002) or prior silencing of β-catenin with siRNA blocked 5-HT-induced PPAR γ reduction and PASMCs proliferation. In addition, inactivation or lack of GSK-3β or inhibition of proteasome function up-regulated β-catenin protein without affecting its mRNA level and reduced PPAR γ protein expression. Taken together, our study indicates that 5-HT suppresses PPAR γ expression and stimulates PASMCs proliferation by modulating GSK-3β/β-catenin axis, and suggests that targeting this pathway might have potential value in the management of PH. Pulmonary hypertension (PH) is a common clinical syndrome characterized by sustained elevation of pulmonary vascular resistance and increased pulmonary arterial pressure, leading to right heart failure and ultimately death . All typPeroxisome proliferator-activated receptor γ (PPAR γ) is a member of the PPAR nuclear hormone receptor superfamily. Besides its metabolic actions , emerginThe Wnt/β-catenin signaling pathway controls many biological processes including cell proliferation, apoptosis and differentiation . β-catenP < 0.01). Figure P < 0.01), indicating that 5-HT effectively stimulates PASMCs proliferation.To examine the effect of 5-HT on PASMCs proliferation, dose-response and time-course of 5-HT on cell proliferation were investigated. Cell proliferation was determined by BrdU incorporation assay. As shown in Figure P < 0.01). Figure P < 0.01). These results suggest that 5-HT suppresses PPAR γ expression in PASMCs.To clarify whether 5-HT reduces PPAR γ expression in PASMCs, cells were treated with different concentration of 5-HT at different time point and the protein level of PPAR γ was determined using Western blotting. As indicated in Figure P < 0.01), and phosphorylation of GSK-3β dropped from 1.99-fold over control in 5-HT-treated cells to 1.09-fold over control in Ly294002 and 5-HT-treated cells (P < 0.01). Figure P < 0.01), while pre-incubation of cells with Ly294002 reduced β-catenin protein level to 1.23-fold over control in response to 5-HT (P < 0.01 versus 5-HT-treated cells). Furthermore, the presence of Ly294002 dramatically blocked 5-HT-induced PPAR γ protein reduction, which increased from 0.5-fold over control in 5-HT-treated cells to 0.87-fold over control in Ly294002 and 5-HT co-treated cells . These results suggest that 5-HT inactivates GSK-3β, up-regulates β-catenin and reduces PPAR γ expression via PI3K/Akt pathway.It has been shown that activation of PI3K/Akt signaling pathway phosphorylates GSK-3β at Ser 9 resulting in inactivation of GSK-3β and subsequent inhibition of β-catenin degradation in several types of non-pulmonary arterial smooth muscle cells , 20. To P < 0.01), while non-targeting siRNA did not affect GSK-3β protein level. Figure P < 0.01).Studies have shown that β-catenin stability and consequent its overall amount in cell is finely regulated by GSK-3β and proteasome system in mammalian cells , 22. To P < 0.01). Suppression of proteasome function by MG132 also reduced PPAR γ protein level, which decreased to 0.67-fold compared with control (P < 0.05). Together, these results suggest that inhibition of GSK-3β or proteasome function leads to accumulation of β-catenin protein and reduction of PPAR γ protein.To examine whether inactivation/loss of GSK-3β or suppression of proteasome function results in PPAR γ reduction in PASMCs, cells were incubated with 1 μM 5-HT for 24 h or transfected with GSK-3β siRNA for 48 h or treated with 10 μM MG132 for 24 h, the protein level of PPAR γ was determined using Western blotting. Figure P < 0.01), while non-targeting siRNA did not affect β-catenin protein level. Figure P < 0.01 versus control), while loss of β-catenin reversed 5-HT-induced PPAR γ reduction, which increased from 0.50-fold over control in 5-HT-treated cells to 0.89-fold over control in 5-HT-treated cells with prior knockdown of β-catenin (P < 0.01). These results suggest that up-regulation of β-catenin particularly mediates 5-HT inhibition of PPAR γ expression in PASMCs.To verify whether up-regulation of β-catenin specifically mediated 5-HT-induced PPAR γ reduction in PASMCs, cells were first transfected with β-catenin sequence specific siRNA for 24 h and then stimulated with 1 μM 5-HT for an additional 24 h, PPAR γ protein level was measured using immunoblotting. As shown in Figure P < 0.01). In addition, prior treatment of PASMCs with pioglitazone also suppressed 5-HT-induced cell proliferation, BrdU incorporation rate declined from a 1.59-fold increase over control in 5-HT-treated cells to a 1.16-fold increase over control in pioglitazone and 5-HT co-treated cells (P < 0.01). These results suggest that post-transcriptional up-regulation of β-catenin and subsequent PPAR γ reduction mediates 5-HT-induced PASMCs proliferation.To clarify whether accumulation of β-catenin by GSK-3β inactivation and subsequent PPAR γ reduction are involved in 5-HT-induced PASMCs proliferation, cells were pre-incubated with 25 μM Ly294002 for 30 min or prior transfected with β-catenin siRNA for 24 h or pre-treated with 10 μM pioglitazone for 30 min and then stimulated with 1 μM 5-HT for an additional 24 h, the proliferation of cells was determined by BrdU incorporation assay. As shown in Figure In this study, we have shown that 5-HT causes the reduction of PPAR γ expression in primary cultured PASMCs, this effect is coupled to the phosphorylation of Akt and inactivation of GSK-3β and subsequent post-transcriptional up-regulation of β-catenin, which further suppresses the expression of PPAR γ and leads to PASMCs proliferation. Our study provides novel molecular insight for 5-HT down-regulation of PPAR γ and stimulation of PASMCs proliferation.Insufficiency of PPAR γ has been shown to be associated with the development of a variety of cancer and other human diseases, while activation of PPAR γ suppresses both tumor and normal cell proliferation –25. ReceWnt/β-catenin pathway plays an essential role in cell fate decision . NumerouGSK-3β is a serine/threonine protein kinase that regulates cell proliferation and apoptosis through multiple intracellular signaling pathways , 39. GSK2 humidified incubator. PASMCs were passaged using 0.25% trypsin  till reaching 80% confluence. All experiments were performed using cells between passages 3 and 6. The purity of PASMCs was determined by immunostaining with α-smooth muscle actin  as described previously. Fluorescence microscope images indicated that cells contained more than 90% of PASMCs (data not shown here). Before each experiment, cells were incubated in 1% FBS-DMEM overnight to minimize serum-induced effects. 5-HT (Sigma) was used to stimulate cell proliferation. Ly294002 (Sigma) was applied to inhibit PI3K activity. MG132  was used as a selective proteasome inhibitor. Pioglitazone  was used to stimulate PPAR γ activation.Primary PASMCs from main pulmonary arteries were prepared from Sprague-Dawley rats (70–80 g) according to the method reported by Wu et al . All ani2 in a humidified incubator. Effects of siRNA transfection were analyzed using immunoblotting.To silence the expression of GSK-3β or β-catenin protein, PASMCs were transfected with sequence-specific or non-targeting control siRNA  using Lipofectamine™ 2000 reagent (Invitrogen) according to the manufacture's protocols. Briefly, PASMCs were seeded into 6-well plates and cultured till reaching 30-40% confluence; 100 nM siRNA and 5 μL Lipofectamine were diluted in 250 μL DMEM, separately, and incubated for 5 min at room temperature. Diluted siRNA was mixed with diluted Lipofectamine and incubated at room temperature for 20 min. Then, the complex of siRNA and Lipofectamine was added into cells and cells were cultured for 48 h at 37°C, 5% CO3 cells per well, allowing to adhere for at least 24 h, and serum starved overnight (1% FBS in DMEM) before the start of experiments. After different treatments, BrdU labeling reagent was added to the wells and incubated for 2 h at 37°C. Cells were then denatured with FixDenat solution for 30 min, and incubated with anti-BrdU mAbs conjugated to peroxidase for 90 min at room temperature. After incubation, antibody conjugate was removed and substrate solution was added for reaction for 10 min. Finally, the reaction product was quantified by measuring the absorbance at 370 nm using a microplate reader . The blank corresponded to 100 μL of culture medium with or without BrdU.To determine PASMCs proliferation, the rate of BrdU incorporation was examined using BrdU ELISA Kit  following the established protocol. PASMCs were seeded on 96-well plates at 5×10Total RNA was extracted from primary cultured PASMCs with Trizol (Invitrogen) following the manufacturer's instructions. Expression of β-catenin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined using Real-time PCR. Complementary DNA (cDNA) was synthesized with the cDNA Synthesis Kit . Real-time PCR was carried out on an IQ™5 Real-time PCR Detection System (Bio-Rad) using SYBR premix Ex Taq II (Takara) as fluorescent dye. The primers for β-catenin  were: forward 5′-CCACGACTAGTTCAGCTGCTTGTAC-3′ and reverse 5′-ACTGCACAAACAGTGGAATGGTATT-3′. Primers for GAPDH (Sangon Biotech) were: forward 5′-GCTGAGTATGTCGTGGAGT-3′ and reverse 5′-GTTCACACCCATCACAAAC-3′. The PCR amplification was performed at 95°C for 1 min, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s and 72°C for 30 s. Expression level of β-catenin was presented as fold increase over controls after normalization with GAPDH.The cultured cells were washed twice with ice-cold PBS and then lysed in RIPA lysis buffer containing 50 mM Tris/HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF and proteinase inhibitors. Lysates were centrifuged at 13,000 rpm at 4°C for 15 min, and the supernatant was collected as sample protein. Protein was separated on SDS-PAGE gel and transferred to a nitrocellulose  membrane via semidry transfer. The NC membrane was then blocked with 5% (w/v) nonfat dry milk in PBS containing 0.1% (v/v) Tween-20. Polyclonal or monoclonal antibodies against phosphor-Akt, total-Akt, phosphor-GSK-3β, total-GSK-3β, β-catenin , PPAR γ  as well as GAPDH  were used according to the manufacturer's protocols. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibodies . Reactions were visualized with SuperSignal West Pico Chemiluminescent Substrate  and then exposed to the autoradiographic film. Signaling was quantified from scanned films using Quality One software (Bio-Rad). Band quantification of aimed proteins was standardized by GAPDH or related total protein.Statistical analysis was carried out using the SPSS 13.0 software. All values are presented as mean ± standard deviation (S.D.). Data were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc test. Probability values of P < 0.05 were considered to represent a statistically significant between groups.


Both epoxy-encapsulated TiO2 nanotubes and amine-immobilized mesoporous silica were incorporated into epoxy and subsequently coated on a carbon steel substrate. The encapsulated TiO2 nanotubes was quantitatively estimated using a ‘dead pore ratio’ calculation. The morphology of the composite coating was studied in detail using transmission electron microscopic (TEM) analysis. The self-healing ability of the coating was monitored using electrochemical impedance spectroscopy (EIS); the coating recovered 57% of its anticorrosive property in 5 days. The self-healing of the scratch on the coating was monitored using Scanning Electron Microscopy (SEM). The results confirmed that the epoxy pre-polymer was slowly released into the crack. The released epoxy pre-polymer came into contact with the amine immobilized in mesoporous silica and cross-linked to heal the scratch.The potential of inorganic nanomaterials as reservoirs for healing agents is presented here. Mesoporous silica (SBA-15) and TiO The self-healing concept has been studied to develop effective corrosion protection coatings for metals123478921112et al.The self-healing in epoxy systems based on the encapsulation of the epoxy and the curing agent in containers provides a repair system that shares chemical properties with the host epoxyInorganic particles with nanocavities have a large surface area, a high pore volume and a low density and stability favorable for the storage of the healing agents. Additionally, the use of inorganic nanomaterials as reservoirs for healing agents can eliminate the tedious encapsulation process. Previously, inorganic particles such as cerium molybdate131820211323242 nanotubes and mesoporous silica as containers to store epoxy pre-polymer and the amine curing agent, respectively. A simple method for the preparation of self-healing epoxy coating for carbon steel by utilizing both mesoporous silica and TiO2 nanotube was used in this study. TiO2 nanotubes (TNT) prepared by a hydrothermal method were used to encapsulate the epoxy pre-polymer. At the same time, amine curing agent was immobilized in high surface area mesoporous silica (SBA-15). The encapsulation of the epoxy monomer in the TiO2 nanotubes and the distribution of mesoporous silica on the composite coating of the filled TiO2 nanotubes were investigated using TEM analyses. The self-healing ability of this two-component self-healing epoxy coating was evaluated using electrochemical impedance spectroscopy (EIS) and Scanning Electron Microscopy (SEM).This research aims to explore the potential of TiO® P-123) with an average Mn-5,800 supplied by Sigma–Aldrich was used as template for the synthesis of mesoporous silica. Tetraethyl orthosilicate (TEOS) (reagent grade 98%) from Sigma–Aldrich was used as the silica source. TiO2 (anatase) powder supplied by Sigma-Aldrich was used as the precursor for TiO2 nanotubes. Hydrochloric acid (HCl), ethanol and sodium hydroxide pellets used were supplied by Sigma–Aldrich.Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (EO20PO70EO20) , purchased from Miller-Stephenson Chemical Co., USA, were used for coating. Epon 815C is a form of Epon 826 diluted with butyl glycidyl ether and was inserted inside the TiOThe morphology of the synthesized nanomaterials was studied using scanning electron microscopic (SEM) and transmission electron microscopic (TEM) techniques. Nova NanoSEM field emission scanning electron microscopy with an accelerating voltage of 5.0 kV was used. The TEM study was conducted using an FEI TECNAI GF20 S-TWIN electron microscopy instrument. The sample was prepared by dispersing the final powders in water and dropping the dispersion onto carbon copper grids. The morphology of the epoxy composite coating was also studied using TEM. Approximately 100-nm-thick sections were microtomed at room temperature using a diamond knife and transferred to carbon-Cu grids.Brunauer–Emmett–Teller (BET) surface area analysis of the nanomaterials was performed using the Micromeritics Chemisorb 2750 pulse chemisorption system. The sample mass was approximately 0.04 g. The sample was degassed in the presence of pure nitrogen for 30–45 min at 200 °C prior to analysis. The BET adsorption/desorption isotherm was determined by nitrogen sorption from a mixture of helium (70%)/nitrogen (30%) gas at the temperature of liquid nitrogen (77 K).The wettability of the coated carbon steel was evaluated using water contact angle measurements. The contact angle measurements were conducted in a Kruss G40 contact angle goniometer at room temperature following the sessile drop principle. Electrochemical impedance spectroscopic (EIS) studies of the coatings were conducted using a Gamry Reference 600 Potentiostat/Galvanostat/ZRA. An Ag/AgCl electrode was used as the reference electrode with a stainless steel counter electrode arranged parallel to the exposed sample to complete the cell. A 3.5 wt% NaCl aqueous solution was used as the electrolyte solution. The sample was in contact with the electrolyte solution, and the measurements were performed at ambient temperature. EIS measurements were performed over the frequency range of 0.01–100,000 Hz. SEM images of the scratched coating surfaces were taken at defined intervals using Benchtop scanning electron microscopy (JCM 6000 – Jeol) to observe the self-healing process.2/g.SBA-15 mesoporous silica was synthesized using the amphiphilic triblock copolymer poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (EO20PO70EO20) as described in ref. 2 nanotubes2 powder was mixed with 20 ml of 10 N NaOH solution in a Teflon beaker and vigorously stirred for 15 min. The mixture was then transferred to a Teflon-lined autoclave and heated in a preheated oven at 130 °C for 10 h. The obtained precipitate was washed with distilled water. The washed precipitate was dipped in 0.1 M HCl solution for 30 min and washed again with HCl solution and distilled water until a solution pH of 7 was achieved. Finally, the synthesized powder was dried. The SEM and TEM images of the prepared TiO2 nanotubes are shown in 2 nanotubes is 173.93 m2/g.A hydrothermal method was used to synthesis TiO2 nanotube. During this process, the air trapped inside the tube lumen was replaced by the epoxy pre-polymer. A schematic illustration of insertion of epoxy monomer into the TiO2 nanotube cavity is shown in Epon815C/15 phr TNT solution was prepared by stirring for 30 min at 1000 rpm. The obtained homogenous solution was placed inside a vacuum jar for 24 h to complete the encapsulation of the epoxy inside the TiOThe prepared mesoporous silica was stirred with an excess of Epikure 3223. The mixture was incubated for 24 h at 25 °C with moderate shaking to immobilize the amine molecules in pores of SBA-15. The immobilization of amine curing agent in the pores of SBA-15 is shown in The epoxy encapsulated Epon815C/15 phr TNT solution was diluted with Epon 826 to make epoxy/7.5 phr TNT solution. This homogenous solution was then mixed with 0.8 phr curing agent immobilized in mesoporous silica. The encapsulated TNT to curing agent immobilized mesoporous silica ratio was fixed based on stoichiometric ratio of epoxy to curing agent used in this study. The stoichiometric ratio of epoxy: diethylene triamine curing agent is 100:11 (For 100 g epoxy 11 g of curing agent). Similarly, the encapsulated TNT: curing agent immobilized mesoporous silica ratio was fixed as 7.5 phr: 0.89 phr. Next, 11 phr curing agent was added to the mixture and was degassed by sonication for 10 min. The mixtures were then coated on one side of the carbon steel to obtain a coating with an average thickness of 300 μm. The coating was allowed to cure for 24 h. The control epoxy coatings were prepared without TNT and mesoporous silica. The control epoxy coating is abbreviated as ‘EP’ and the epoxy coating loaded with epoxy pre-polymer encapsulated TNT and curing agent immobilized mesoporous silica is denoted as ‘EP/TNT/Imeso’. The abbreviated names are used for the coating samples in the subsequent text.et al.282 nanotube loaded with the epoxy monomer. In this approach, the degree of nanotube filled with the epoxy monomer in the composite was calculated in terms of ‘dead pore ratio’ using the 2 nanotube (7.5 phr), the presence of mesoporous silica is not considered while calculating the dead pore ratio.The approach adopted by Suzuki The vacant volume (V) (not filled with monomer) per weight (g) of loaded TNT in the EP/TNT/Imeso composite coating is given by0 is the total pore volume of the TNT (0.2784 cm3/g), X is the additive amount ratio in wt % of the TiO2 nanotube in the composite (6.3291), and ρ, ρ (epoxy) and ρ(TNT) are the density of the composite, epoxy polymer and TNT, respectively. (ρ(TNT) − 4.23 g/cm3, ρ(epoxy) −1.1983 g/cm3 and ρ −1.2775 g/cm3).where VThe calculated dead pore ratio for the EP/TNT/Imeso coating composite was 94%. A 100% dead pore ratio represents that all the nanotube pores are filled with the polymer, and a 0% dead pore ratio indicates that all the pores are empty. A 94% dead pore ratio value of the EP/TNT/Imeso coating composites indicates that the pores are satisfactorily filled with epoxy.2 nanotubes  and unhealed scratched (EP/TNT/Imeso with scratch_0 h and EP/TNT/Imeso with scratch_24 h) coatings. The impedance value in the middle frequency region is higher for the scratched EP/TNT/Imeso coatings undergoing the healing process  than that of the unscratched and unhealed scratched EP/TNT/Imeso coatings, indicating a high charge-transfer resistance of the healed coatings.The self-healing ability of the coatings was evaluated using an EIS study of the scratched samples at regular time intervals3Further, the Nyquist plots for the scratched coating from 0 to 96 hours of immersion time are shown in The electrical equivalent circuit used to fit the EIS plots for the control epoxy coating and the EP/TNT/Imeso coating is shown in 2 nanotube into the scratch and its successive crosslinking upon contact with the immobilized amine in mesoporous silica. The larger size of the mesoporous silica facilitates more chance of contact with the epoxy pre-polymer released from the TiO2 nanotube upon crack.The self-healing ability of the EP/TNT/Imeso coating was monitored using SEM. 2 nanotubes (TNT) with an average tube diameter of 10 nm were synthesized and effectively used as containers to load an epoxy pre-polymer and an amine curing agent. The epoxy pre-polymer was encapsulated inside the lumen of TiO2 nanotubes by a vacuum loading process. The amine curing agent was immobilized in mesoporous silica by shaking incubation. Epoxy coatings with both epoxy-encapsulated TiO2 nanotubes and amine immobilized mesoporous silica were prepared on carbon steel. The satisfactory filling of the TiO2 pores was confirmed by the estimated dead pore ratio of 94% for EP/TNT/Imeso coating composites. TEM images of the EP/TNT/Imeso coating composites showed the localization of the amine curing agent immobilized mesoporous silica among the groups of filled TiO2 nanotubes. The self-healing ability of EP/TNT/Imeso was confirmed by the EIS bode plots. EP/TNT/Imeso coating recovered 57% of its anticorrosive property in 5 days. The experimental EIS data were well-fitted with electrical circuits, and the subsequently extracted parameters were further used to confirm the self-healing ability of the EP/TNT/Imeso coating. The increase in coating resistance (Rpo) with the immersion time suggested the healing of the scratch in the EP/TNT/Imeso coatings. The self-healing process was monitored using SEM. The self-healing of the EP/TNT/Imeso coating has been proposed to occur by the release of the epoxy pre-polymer from the TiO2 nanotubes and its further crosslinking with the amine curing agent immobilized in the mesoporous silica. The slow and steady recovery of the damage makes this coating suitable for long term corrosion resistance applications. This self-healing technology could be extended to epoxy composites used in structural and aircraft parts. Further studies of the detailed kinetics of the healing process are in progress.Mesoporous silica (SBA 15) with a high surface area and an average pore size of 4 nm and TiOHow to cite this article: Vijayan P., P. and Al-Maadeed, M. A. S. A. TiO2 nanotubes and mesoporous silica as containers in self-healing epoxy coatings. Sci. Rep.6, 38812; doi: 10.1038/srep38812 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


MALAT1) is a long non‐coding RNA, broadly expressed in mammalian tissues including kidney and up‐regulated in a variety of cancer cells. To date, its functions in podocytes are largely unknown. β‐catenin is a key mediator in the canonical and non‐canonical Wnt signalling pathway; its aberrant expression promotes podocyte malfunction and albuminuria, and contributes to kidney fibrosis. In this study, we found that MALAT1 levels were increased in kidney cortices from C57BL/6 mice with streptozocin (STZ)‐induced diabetic nephropathy, and dynamically regulated in cultured mouse podocytes stimulated with high glucose, which showed a trend from rise to decline. The decline of MALAT1 levels was accompanied with β‐catenin translocation to the nuclei and enhanced expression of serine/arginine splicing factor 1 (SRSF1), a MALAT1 RNA‐binding protein. Further we showed early interference with MALAT1 siRNA partially restored podocytes function and prohibited β‐catenin nuclear accumulation and SRSF1 overexpression. Intriguingly, we showed that β‐catenin was involved in MALAT1 transcription by binding to the promotor region of MALAT1; β‐catenin knock‐down also decreased MALAT1 levels, suggesting a novel feedback regulation between MALAT1 and β‐catenin. Notably, β‐catenin deletion had limited effects on SRSF1 expression, demonstrating β‐catenin might serve as a downstream signal of SRSF1. These findings provided evidence for a pivotal role of MALAT1 in diabetic nephropathy and high glucose‐induced podocyte damage.Metastasis associated lung adenocarcinoma transcript 1( Long noncoding RNAs (lncRNAs) are non‐protein‐coding transcripts longer than 200 nucleotides and have emerged as important regulators in a variety of cellular responses, developmental and disease processes via epithelial‐mesenchymal transition (EMT) mechanism in oral squamous cell carcinoma (OSCC), when both NF‐κB and β‐catenin were triggered nuclear translocation Prior studies showed MALAT1 acted as an important oncogene in numerous cancers Mature MALAT1 is localized in the cell nuclei with a cytoplasmic tRNA‐like small RNA, known as mascRNA via down‐regulating SRSF1 overexpression, a MALAT1 lncRNA‐binding protein, and partial reversal of β‐catenin nuclear accumulation triggered by high glucose. In addition, we showed MALAT1 levels were also under the regulation of β‐catenin via its binding ability to MALAT1 promotor region; β‐catenin gene knock‐down led to decreased MALAT1 levels, demonstrating a novel feedback regulation between MALAT1 and β‐catenin.In this study, we provided evidence that MALAT1 lncRNA was dysregulated in STZ‐induced diabetic nephropathy when proteinuria was marked and involved in high glucose‐induced podocyte damage. MALAT1 knock‐down rectified podocyte damage Antibodies specific for β‐catenin and SRSF1 were purchased from Abcam . Anti‐p‐cadherin antibodies purchased from Santa Cruz Biotechnology, Inc. . Anti‐ZO‐1 antibodies were purchased from Thermo Fisher Scientific Inc. . Anti‐desmin antibodies were from Immunoway. Anti‐β‐actin antibodies were obtained from Proteintech . Secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. . Dylight 594 and 488 conjugated secondary antibodies were from Abcam .ad libitum; urine was collected for the determination of urinary protein when the mice were weighed and placed in metabolic cages for 24 hrs. At the end of 12 weeks after the onset of diabetes, mice were killed, and the kidneys were harvested for the following experiments.A total of 20 male C57BL/6 mice were purchased from the Animal Center Affiliated to Shandong University. All animal studies were carried out with the review and approval of the animal care and use committee of Shandong University. Mice were randomly allocated into two groups, either intraperitoneal injected with STZ dissolved in a low pH (pH 4.5) citrate buffer (50 mg/kg for 5 consecutive days) or citrate buffer alone. Hyperglycaemic state was monitored and maintained on a standard rodent diet with water via Professor Jie Ding . In brief, podocytes were cultured on type I collagen in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) , 100 U/ml penicillin and 100 mg/ml streptomycin under permissive conditions 33°C plus 10 U/ml mouse recombinant γ‐interferon . Cell differentiation was induced by maintaining podocytes on type I collagen at 37°C without γ‐interferon under non‐permissive conditions for at least 14 days Conditionally immortalized mouse podocytes were kindly provided by Professor Peter Mundel  5 cells/well in a 24‐well plate) with the indicated siRNAs using Lipofectamine® RNAiMAX Transfection reagent (Thermo Fisher Scientific Inc.). Real‐time reverse transcriptase‐PCR was used to validate the efficiency of MALAT1 knock‐down. After a 48‐hrs incubation with MALAT1 siRNA or control siRNA, podocytes were then treated with high glucose (30 mM) for an additional 48 hrs. Cells that were not transfected and incubated with low‐glucose (5.6 mM) medium for 48 hrs were the controls. The lentiviral shRNA constructs targeting mouse β‐catenin (5′‐GCTGCGGAAGATGGGATCAAA‐3′) and a scrambled shRNA (5′‐GACTTCATAAGGCGCATGC‐3′), as well as lentiviral expression vectors for murine β‐catenin, and empty control vectors were purchased from Cyagen , and lentivirus infection of the vectors was performed following the protocol provided by the manufacturer. Briefly, viral supernatants were added for 24 hrs to infect mouse podocytes. And geneticin (250 mg/ml) was added into the culture medium to select cells with stable viral integration.MALAT1 siRNAs  and a negative control sequence (control siRNA) were purchased from Invitrogen (Thermo Fisher Scientific Inc.). Transfection of podocytes was performed . Briefly, after reverse transcription of total RNA, cDNA was used as a template for the PCR reactions using gene‐specific primer pairs. Amplification was performed using SYBR® Premix Ex Taq™ Kit  in the LightCycler® 480 Real‐Time PCR system . The primers were purchased from Sangon Biotech Co., Ltd . The sequences were designed as follows  membranes; the membranes were then blocked with 5% milk or BSA for 1 hr and incubated at 4°C overnight with primary antibodies against the following target proteins: ZO‐1 (1:1000), p‐cadherin (1:1000), desmin (1:1500), β‐catenin (1:1000), SRSF1 (1:1000) and β‐actin (1:1000). The membranes were then washed three times with TBST for 5 min. and incubated with species‐specific peroxidase‐conjugated secondary antibodies diluted in blocking buffer for 1 hr at room temperature. Specific bands were detected using the ECL system and the Bio‐Rad electrophoresis image analyser .2 and 1 μM ZnCl2 [pH 7.6]) for 40 min. followed by incubation in developing buffer  at 37°C for 36 hrs. Gels were stained with 0.05% bright blue R‐250 for 3 hrs, and then washed with distaining solution (methanol: acetic acid: water 30:10:60) and quantified using NIH image.MMP‐2 enzyme activity of the podocytes under different conditions was monitored by gelatin zymography. Briefly, supernatants from stimulated or control cells were collected and centrifuged and were mixed with 4 × non‐reducing loading buffer . Protein separation was performed by electrophoresis using 10% polyacrylamide SDS gel containing 1 mg/ml gelatin, after which the gels were denatured in 2.5% Triton X‐100 for 45 min., equilibrated in buffer  for 1 hr at room temperature to block non‐specific binding. Immunostaining was performed with appropriate primary antibodies at 4°C overnight and Dylight 594 or 488‐conjugated IgG at room temperature for 1 hr for visualization. 4′, 6‐diamidino‐2‐phenylindole (DAPI) was used to visualize the nuclei. Images were observed and captured on an inverted phase/fluorescence microscope .in Situ Hybridization kit were purchased from Ribio . Briefly, cultured podocytes under different conditions were plated onto different 6‐well plates and fixed in 4% paraformaldehyde for 10 min., followed by ice‐cold PBS containing 0.5% Triton X‐100 for 5 min., and then incubated with pre‐hybridization buffer for 30 min. at 37°C to block non‐specific binding. Hybridization buffer was preheated in a 37°C water bath, and the MALAT1 FISH probe working buffer was prepared by diluting probe stock solution in hybridization buffer (1:40). Cells were incubated with probe working buffer at 37°C overnight and followed by DAPI staining to visualize the nuclei. Images were observed and captured on an inverted phase/fluorescence microscope .MALAT1 probe and fluorescence g at 4°C for 5 min. For sonication, nuclei pellets were re‐suspended in 500 μl RIP Cross‐linked Lysis Buffer containing protease/RNase inhibitors and sonicated on wet ice using a sonicator . Antibody‐beads complex were prepared, and 50 μl sheared cross‐linked chromatin was incubated with antibody‐beads with rotation overnight at 4°C. Cross‐link reversal was performed by incubating the beads in 200 μl elution buffer containing 10% SDS and proteinase K with shaking at 60°C for 30 min. RNA extraction was described previously RNA‐binding protein immunoprecipitation was performed using Magna RIP kit . In brief, podocytes or HEK‐293 cells were fixed by 0.3% formaldehyde and quenched with glycine, and scraped to pellet cells, which were re‐suspended and collected by high speed vortex and spinning at 800 7 podocytes with or without catenin gene knock‐down or HEK‐293 cells were fixed with 1% PFA, quenched with glycine, washed with cold PBS and re‐suspended in nuclei isolation buffer with a protease inhibitor cocktail. The chromatin was sonicated on wet ice to obtain chromatin fragment lengths between 200 and 1200 bp. The protein‐DNA complexes were incubated with 1 μg of anti‐β‐catenin, or non‐specific IgG antibodies couple with protein A/G beads overnight followed by washing and elution. Cross‐links were reversed at 65°C for 2 hrs and 95°C for 15 min. DNA was purified and subjected (20 ng each) to real‐time PCR analysis.According to the EZ‐Magna CHIP™ HiSens kit , 1 × 10MALAT1 promotor‐luciferase reporter vectors and pGL3‐TK‐Renilla luciferase control vectors for 24 hrs using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) with or without further incubation with either low (5.6 mM) or high‐glucose (30 mM) treatment for 48 hrs. The luciferase activities were analysed using a Dual‐Luciferase® Reporter Kit .The luciferase reporter constructs containing the MALAT1 promoter region were generated by PCR using the primers: sense 5′‐TGGTCCTGGGAGAAAGAGAA‐3′; antisense 5′‐CCTGGGAGGACAGAGGGTAT‐3′. Briefly, cultured podocytes stably overexpressing β‐catenin or control cells were cotransfected with pGL3‐To determine the permeability of albumin influx, differentiated podocytes were seeded on collagen I‐coated Transwell filters  and stimulated either with low glucose or high glucose. After 48 hrs of stimulation, the top chamber was refilled with 200 μl of RPMI 1640 media and the bottom with 600 μl of RPMI 1640 media containing 1.5 mg/ml FITC‐albumin (Solarbio). Cells were then incubated at 37°C, and 30 μl of media from the top chamber were carefully collected at the indicated time‐points. The absorbance of FITC‐albumin was determined at 490 nm with a fluorescence multi‐well plate reader  to indicate the filtration/influx function of monolayer podocytes.t‐test, one‐way anova and LSD‐t test, and two‐way anova. P < 0.05 were considered to be statistically significant.Experiments were performed at least three times. Values were reported as mean ± S.D. Data were analysed using SPSS 19.0 software. Statistical significance was assessed using Student's P < 0.01)  Table .P < 0.01)  (Table P < 0.01)  Table . ImmunofP < 0.01), after which MALAT1 was restored to a level comparable to that in podocytes under low‐glucose conditions (P > 0.05) but notably decreased until 48 hrs of treatment (P < 0.01), showing a trend from rise to decline     Figs A. Immuno01) Figs C and 4B.P < 0.01)      Fig. B. Intrig05) Fig. C, nor an05) Fig. A, compar05) Figs C and 4B.In vivo, we have shown an up‐regulation of SRSF1 mRNA levels. Next, we examined SRSF1 expression in cultured podocytes. Results showed that high glucose resulted in a notable increase in SRSF1 expression on both protein and mRNA levels (P < 0.01), which were significantly decreased by MALAT1 knock‐down, compared with podocytes without transfection (P < 0.05)  or in human 293 cells (P < 0.05)   or human 293 cells (P < 0.01)                  Fig. A. Result01) Fig.  B but al01) Fig. C, sugges01) Fig. D; β‐cate01) Fig. D. Simila01) Fig. D. Next, 05) Fig. A. Using 01) Fig. B. These 01) Fig. C. Notabl01) Fig. C, sugges05) Fig. D; levels01) Fig. D; in cel05) Fig. D; and ov05) Fig. D, implyi05) Fig. E, indicavia its ability to breakdown of extra‐cellular matrix (ECM) and glomerulus in vivo and in vitro data provided evidence that MALAT1 dysregulation played a key role in diabetic podocyte injury and proteinuria; imbalanced MALAT1 levels contributed to disruptions of the integrity of podocyte architecture and function; preserved SD structure and thus alleviated actin interference might be one of the major consequences podocytes benefited from MALAT1 deletion.MALAT1 is a highly conserved mRNA‐like long non‐coding RNA (lncRNA), originally considered as a pivotal prognostic factor in various metastatic carcinomas via recognition of β‐catenin pre‐mRNAs, and thereby altered at protein levels. Intriguingly, MALAT1 levels were affected by β‐catenin as well through binding to the promotor region of MALAT1. β‐catenin shRNA transduction led to decreased mutual binding and thus diminished MALAT1 expression; overexpression of β‐catenin, on the contrary, was able to increase promotor activity of MALAT1. These observations suggested that there might exist a feedback loop between MALAT1 and β‐catenin in the podocyte nucleus under normal conditions: β‐catenin regulated MALAT1 transcription via binding to its promotor and regulating the promotor activity; MALAT1 in turn changed the pattern of pre‐mRNA processing of β‐catenin post‐transcriptionally. This loop was damaged by high‐glucose treatment, which resulted in an early increase in MALAT1 that contributed to nuclear accumulation of β‐catenin at later stage.Of interest, we showed that MALAT1 knock‐down was able to reduce the expression of total protein and mRNA levels of β‐catenin. Under normal conditions MALAT1 was highly expressed and retained in the cell nucleus of podocytes. Such localization is suggestive of a potential role in the organization or regulation of gene expression It was notable that in our observations the nuclear‐translocated β‐catenin did not significantly increase MALAT1 levels despite its binding ability to MALAT1 promotor and its enhancement in MALAT1 promotor activity, even in cells overexpressing β‐catenin, where MALAT1 levels were not significantly higher than but only comparable to low‐glucose counterparts, suggesting that MALAT1 might be as well under the control of some other molecules post‐transcriptionally after high‐glucose stimulation. Many ncRNAs were implicated in regulating RNA processing either directly through base pairing or indirectly through protein intermediates in vivo data further identified a concomitant elevation of SRSF1 mRNA and MALAT1 in the kidney of STZ‐induced diabetic mice, suggesting MALAT1 and SRSF1, with associated disordered pre‐mRNA processing, contributed to the development and progression of diabetic nephropathy. Prior studies suggested that lncRNA MALAT1 was raised in the retinas of STZ‐induced diabetic rats, blockade of which alleviated retinal inflammation via reducing expression of ICAM, VEGF and TNF‐α Our in vitro that β‐catenin knock‐down resulted in a further extent of MALAT1 down‐regulation, whereas SRSF1 protein expression was not significantly altered, suggesting that β‐catenin might serve as a downstream effector of SRSF1 after high‐glucose treatment. By shuttling between the nucleus and the cytoplasm, SRSF1 interacted with and activated mTOR to promote translation via preventing early up‐regulation of MALAT1, that was, the initial key event in the challenge of high glucose.Next, we were interested to find et al. and our previous studies illustrated that EMT was a potential mechanism leading to podocyte malfunction Growing evidence has illustrated that MALAT1 played a negligible role in epithelial‐mesenchymal transition (EMT) of diverse cancer cells via changing the pattern of alternative splicing of gene targets through its physical binding to MALAT1; inhibition of MALAT1 broke the cascade and rectified podocytes dysfunction stimulated by high glucose (Fig. In summary, we demonstrated that MALAT1 misregulation was involved in the progression of diabetic nephropathy, and we showed a potential feedback loop between MALAT1 and β‐catenin playing a role in high glucose‐associated podocyte impairment, with SRSF1 as a important participant potentially ose Fig. . These oThe authors declare that there is no conflict of interest associated with this manuscript.


Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/β-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/β-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous β-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/β-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD. At the functional level, Nlgns enhance the formation and maturation of specific types of synapses. For instance, while Nlgn1 is found preferentially in excitatory and Nlgn2 in inhibitory and cholinergic synapses,27 Nlgn3 localizes at both excitatory and inhibitory synapses.32 Nlgn4X has been observed in inhibitory glycinergic synapses.33 Currently, mice expressing Nlgn3 mutated forms display autism-like behaviors and are highly sought as experimental models for the study of ASD pathology.35Neuroligins (Nlgns) are postsynaptic cell-adhesion molecules that act as ligands for presynaptic Neurexins (Nrxns).36 This hypothesis has received considerable attention recently,40 mainly since it has been observed that 39% of the more disruptive de novo mutations in ASD family trios were found within interconnected networks containing chromatin remodeling, synaptic, and Wnt/β-catenin signaling genes.42 Likewise, integration of RNA-seq expression profiles during brain development with protein–protein interaction networks have identified highly modules enriched of connected Wnt signaling genes associated with ASD.43 At the genetic level, several Wnt/β-catenin components have been associated with ASD, including the canonical Wnt2 ligand,44 Wnt/β-catenin target genes Engrailed 2 (EN2)45 and the hepatocyte growth factor receptor (MET),48 and cadherins encoding genes such as CDH5, CDH8, CDH9, CDH10, CDH13, CDH15, PCDH10, PCDH19, and PCDHb4,49 some of which interact with β-catenin in cell-cell adhesion complexes. More recently, the chromo-helicase domain protein 8 (CHD8),51 which inhibits β-catenin through direct binding,52 and DYRK1A that modulates Wnt/β-catenin signaling through interaction with the p120 catenin,53 have been found to be associated with ASD. Interestingly, CHD8 and DYRK1A harbor recurrent disruptive mutations and are highly correlated with head size abnormalities,51 a feature commonly observed in ASD. Moreover, rare de novo genetic variants in the β-catenin (CTNNB1) gene itself have been implicated in severe intellectual disability.54We predicted earlier that sustained gain-of-function of Wnt/β-catenin signaling in the developing brain could be responsible for the onset/development of ASD and that this effect involves the additive effect of genetic variants within components and/or genes whose products modulate its functional activity.57 First, in utero exposure to teratogens such as valproic acid  causes a higher incidence of ASD in the offspring59 and VPA is known to increase cytosolic and nuclear β-catenin levels and activate Wnt/β-catenin-dependent gene expression, by a complex mechanism involving inhibition of histone deacetylase (HDAC) and glycogen synthase kinase-3α/β (GSK3α/β) activities.61 Second, in Tuberous sclerosis the tumor suppressor complex formed by hamartin (TSC1 in Chr 9) and tuberin (TSC2 in Chr 16) interact with the β-catenin degradation complex and thus modulates the action of Wnt/β-catenin signaling.63 Finally, several inflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interferon γ (IFN-γ), are elevated in peripheral blood cells, serum, plasma, cerebrospinal fluid, or in the brains of ASD children.72 In this context, recent evidence indicates that Wnt/β-catenin and non-canonical Wnt signaling have both pro- and anti-inflammatory activity73 and that the signaling cascade is involved in inflammation-driven brain damage and inflammation-directed brain repair.74 Wnt3a selectively increases the expression of proinflammatory immune response genes in microglia and enhances the release of de novo IL-6, IL-12, and TNF-α.75 Nevertheless, the transcriptional program elicited by Wnt/β-catenin signaling in different types of brain cells has received little attention.79Further support for a role of Wnt/β-catenin signaling in ASD comes from pharmacological studies, research in ASD comorbidities such as Tuberous sclerosis or in the recognition that an abnormal immune response plays an important role in the onset or development of the disorder.81 that others and we have observed that Wnt/β-catenin signaling enhances excitatory neurotransmission in hippocampal neurons,85 and that the transcriptional program controlling Nlgn3 expression and function is currently unknown, here we investigated the functional effects of the signaling cascade on Nlgn3 expression and synaptic function in hippocampal neurons.Therefore, considering that Wnt/β-catenin signaling has an essential role in ASD affected regions such as the frontal cortex and the hippocampal formation,86 Cells were maintained for 14 days in vitro (DIV) on culture plates (3.5 × 105 cells/plate) coated with poly-l-lysine . Primary neurons were grown on Neurobasal medium supplemented with B27  and 50% media was replaced every 3 days. Cells were kept at 37 °C in 5% CO2 incubator and saturated humidity. The study protocol with rat embryos was approved by the Bioethical Committee of Universidad Andres Bello, Chile (026/2013) and was conducted in accordance with the Ethical Guidelines for Treatment of Laboratory Animals of the National Commission on Science and Technology (CONICYT- Chile).Primary hippocampal neurons were obtained from E18 Sprague-Dawley rat embryos  as described.90 , HEK293T, Wnt3a-L-cells mouse fibroblasts and control-L-cells , were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin , 1% P/S  and kept at 37 °C in 5% CO2 incubator with saturated humidity.HT22 mouse hippocampal cells92 The presence of the Wnt3a protein was detected with an anti- Wnt3a antibody . Purity was analyzed by SDS–PAGE (8%), stained with Coomassie Blue G250, and analyzed through densitometry by using software ImageJ.93Wnt3a is a specific Wnt/β-catenin signaling agonist that can be efficiently recovered from conditioned medium from Wnt3a-secreting L-cells (Wnt3a-CM). Wnt3a purification was carried out, as previously described.5 cells per ml) and stimulated with 200–400 ng/ml of purified Wnt3a protein or with 10–20 mM LiCl (Sigma-Aldrich) and collected for mRNA or protein analyses, as described.95Primary cultures of hippocampal neurons or HT22 cells were seeded in 6 well culture plates  and 2 µg of RNA was reverse transcribed with Affinity Script QPCR cDNA synthesis kit . q-PCR was performed in a Stratagene Mx3005P thermal cycler using 40 ng of cDNA, Brilliant II SYBR Green qPCR Master Mix (Agilent Technologies), and 200–400 nM of primers targeting known Nlgn3 mouse and rat mRNA isoforms  or control non-targeting shRNA  were obtained from MISSION® . Lentiviral particles were produced in HEK293T cells co-transfected with the pLKO.1 construct, pCMV-dR8.91 (Delta 8.9) plasmid  and pVSV.G plasmid (at a 3:2:1 ratio). Transfections were performed using Lipofectamine 2000 . Lentiviral particles were harvested 48 h post transfection, filtered through a cellulose acetate filter (0.45 µm) and concentrated by centrifugation  with a 100 kDa Ultra15 Amicon filter . Infections were carried out in HT22 cells plated in 60 mm diameter with 80–90 % confluence for 48 h using 100 µl concentrated virus.Nlgn3, β-catenin and β-actin proteins were analyzed by western blot using the H-55 anti-Nlgn3 antibody , the monoclonal E-5 β- catenin antibody  or with the anti-β-actin antibodies . Membranes were washed (3×) and incubated with appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature, washed and incubated for 2 min with enhanced chemiluminescence solution  and exposed for 1–3 min on Carestream Kodak BioMax films (Sigma-Aldrich). Secondary antibodies were: goat-anti rabbit IgG-HRP , goat-anti mouse IgG-HRP .95 Activity of Nlgn3 promoters was measured in 80–90% confluent HEK293 cells seeded in 24 well culture plates. Cells were co-transfected using Lipofectamine 2000  for 24 h with either pNL3 or pSUPERTOPFlash (STF) luciferase constructs,97 in the absence or presence of constitutively active β-catenin S33Y or the dominant negative ΔTCF4 constructs.98Nlgn3 promoter-luciferase fragments were generated by conventional PCR from genomic DNA extracted from either mouse HT22 hippocampal cells or Sprague-Dawley primary neurons using specific primers containing restriction sites (Supplementary Table The pRL-TK Renilla luciferase plasmid (1 ng) was used as an internal control. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay (Promega) in a Victor-3 multiplate reader . Promoter activity was normalized as the ratio between Firefly and Renilla luciferase units. Site-directed mutagenesis in TBE Sites II and III were generated using the pNL3Mm1.4 construct as background with M1 or M2 primers , LiCl or CHIR 98014 (Sigma-Aldrich), as Wnt/β-catenin pharmacological agonists.100 Cells were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature and then the reaction was stopped with 0.125 M glycine. Samples were sonicated with the M220 Focused-ultrasonicator  using milliTube caps (Covaris), following the high cell chromatin shearing protocol suggested by the manufacturer. In total 25 µg of chromatin was used in each ChIP assay. Endogenous β-catenin bound to TBE Sites within the mouse Nlgn3 promoter was immunoprecipitated with rabbit anti β-catenin antibody . Cross-linked chromatin fragments averaging 200–300 bp were assessed by q-PCR as described above. Antibody specificity was assayed with normal rabbit-IgG .ChIP studies were performed in HT22 neurons, as described.l-lysine (Sigma- Aldrich) coated coverslips (5.0 × 105 cells/plate) were fixed using 4% formaldehyde-4% sucrose (pH: 7.0) solution for 20 min at room temperature. Cells were permeabilized with Triton X-100 0.2 % for 5 min and treated with blocking solution  for 30 min. Primary antibodies against Nlgn3 , PSD-95 , and MAP2   were used and Alexa Fluor Antibodies  for secondary staining. Samples were examined on a LSM780 confocal microscopy . Images were deconvolved using the ImageJ/FIJI101 plugin DeconvolutionLab102 using theoretical point spread functions. Total Nlgn3 fluorescence intensity was measured as arbitrary units in all dendritic processes with clear signal of MAP2 in 15 µm segments that consistently covered an area between 40 and 200 µm2. To illustrate colocalization between Nlgn3 and PSD-95 proteins, three-dimensional isosurfaces of dendritic processes were created using Imaris v8.2  and by applying the ImarisSurface tool  on the MAP2 fluorescent signal. Nlgn3 and PSD-95 clusters inside of dendritic processes were rendered using the ImarisSpots tool. Co-localization coefficients of Nlgn3 and PSD-95 (Pearson and Manders) were determined using the ImarisColoc tool in an average of four dendrites per neuron, which were selected based on brightness103 and length (~15 µm). Briefly, while Pearson’s correlation coefficient is an adimensional parameter that establishes whether colocalization exists independent of signal strength, thus avoiding the possibility of false positives, the Manders coefficient represents directly the percentage of colocalization between two signals.Control and Wnt3a-treated rat hippocampal neurons (14 DIV) cultured in poly-post hoc test. To compare two groups, we performed a two-tailed Mann–Whitney test. Statistically significant p-values are shown as *P < 0.05, **P < 0.01 and ***P < 0.001.Data are presented as mean ± s.e.m. Each experiment was repeated three times with three replicates. All data processing and analysis were completed before unblinding of the analyzer. Sample size was chosen according to previous reports and our pre-experiments. A minimum number of three biological replicates were performed to ensure reproducible and robust changes. No cell samples or animals were excluded from the analysis. Data were analyzed with Prism v5 . For the comparison of more than two groups, we used one-way ANOVA test followed by Kruskal–Wallis 106 most Wnt/β-catenin target genes have regulatory sequences within 2.5 kb clustering around transcriptional start sites (TSS). We searched for core TCF/LEF-binding elements (TBE: CTTTG) known to mediate β-catenin transcriptional activation of Wnt target genes, including a 3000 bp region upstream of the predicted transcriptional start site (TSS) in human, mouse and rat Nlgn3 gene sequences  or with LiCl (10 and 20 mM), which acts as a pharmacological inducer of Wnt/β-catenin signaling.99 We observed that both treatments increased Nlgn3 expression   upon activation of the signaling cascade in different cell lines and primary cultures of hematopoietic cells.ion Fig.  and that 3) Fig.  and that 3) Fig. . In addi 3) Fig. , at a ti 3) Fig. . We conc98 protein  and assayed its activity through transient transfections in HEK293 cells. This promoter region included 2448 bp upstream of the TSS , 87 bp of the unstranslated exon 1 and 314 bp of intron 1, contained nine putative TBE sites Fig.  and dispp < 0.05; Fig. 98 We found that the enhancement induced by β-catenin on pNL3Mm1.4 promoter activity was significantly antagonized ( > 50%) by co-expressing ΔTCF4 for 24 h , containing TBE sites I–IV and basal elements, was significantly augmented in the presence of constitutively active β-catenin (S33Y) co-transfected for 24 h , we introduced through site-directed mutagenesis two-nucleotide changes in each TBE sequence, using pNL3Mm1.4 as the template  assays in HT22 cells. We observed that a significant binding of β-catenin to TBE sites II and III within the mouse Nlgn3 promoter was induced after treatment with either cell-conditioned media containing Wnt3a  for 4 h or 20 mM LiCl for 24 h  Fig. . In addiate Fig. . Remarka32 Given that we reported that purified Wnt3a modulated intracellular calcium and enhanced excitatory neurotransmission in hippocampal neurons,84 we hypothesized that the signaling cascade may enhance synaptic function by increasing Nlgn3 levels in dendritic processes, where it would interact with PSD-95 in the postsynaptic terminal. Therefore, we incubated 14 DIV rat hippocampal neurons in the absence or presence of purified Wnt3a (400 ng/ml) for 2 or 24 h and performed immunofluorescence analyses to examine whether activation of the signaling cascade affects the differential recruitment of Nlgn3 to dendritic processes. First, Nlgn3 was distributed along dendrites in a punctate manner, in agreement to previous reports,32 where it regularly colocalized with PSD-95 protein  under control conditions, and that such interaction was clearly enhanced when cells were incubated in presence of signaling agonists. While our results represent the first functional characterization of the Nlgn3 promoter, we note that Nlgn1 and Nlgn2 have already been predicted by bioinformatics approaches as potential β-catenin target genes.78 Accordingly, activation of the signaling cascade by the chemical compound curcumin has been shown to effectively enhance Nlgn1 expression in endogenous neural stem cells.114 Interestingly, curcumin has shown anti- inflammatory and antioxidant properties and is being considered as a therapeutic option in cancer and various prevalent neurological disorders, including ASD.117An excess of synapses in frontal, temporal and parietal lobes has been found in children with ASD compared to age-matched controls118 axonal remodeling,119 patterning and maturation of functional synapses122 and excitatory neurotransmission.85 Likewise, increased β-catenin levels are critical for synaptic structure and dendritic arborization.125 In this regard, increased Nlgn3 intensity and Nlgn3 co-localization with PSD-95 after 24 h Wnt3a treatment supports the idea that the signaling cascade is involved in excitatory synaptogenesis through recruiting and clustering of proteins in dendritic processes. For instance, while large clusters of PSD-95 are indicative of spine stabilization126 it has been observed that similar treatment of hippocampal neurons with canonical Wnt7a, consistently increased PSD-95 puncta in a time-dependent manner and in a calcium/calmodulin-dependent protein kinase II (CAMKII) dependent mechanism.122 Interestingly, CAMKII activation is dependent of calcium entrance into the synaptic terminal and our group has already shown that Wnt3a increases transient calcium currents.84 Altogether, these data support a regulatory role for this kinase in ASD and other neurodevelopmental diseases.128It is widely accepted that Wnt/β-catenin signaling is essential for brain development and function and is increasingly recognized that the cascade has a central role in ASD neurodevelopmental pathology. Indeed, Wnt/β-catenin signaling is involved in neurogenesis,129 and recent experiments have shown that this neuronal activity involves the shedding of the Nlgn3-extracellular-domain from the postsynaptic surface.130 It would be interesting to examine whether Nlgn3 expression and clustering promoted by the Wnt/β-catenin signaling cascade leads to Nlgn3 shedding and how this process relates to neuroinflammation and defects in excitatory/inhibitory neurotransmission.132Cortical neuronal activity exerts mitogenic effects on neural and oligodendroglial precursor cellsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Table S1


To evaluate the use of ear endoscopy in the postoperative management of open mastoidectomy cavities, and to test whether ear endoscopy improves inspection and cleaning compared with ear microscopy.Prospective study. Thirty-two ears were divided into two groups: group 1, examination and cleaning of mastoid cavities under endoscopic visualization after microscopic standard ear cleaning; group 2, examination and cleaning of mastoid cavities under microscopic visualization after endoscope-assisted ear cleaning. We assessed the ability of each method to provide exposure and facilitate cleaning, comparing the benefits of microscopy and endoscopy when used sequentially and vice-versa.Endoscopy provided additional benefits for exposure in 61.1% of cases and cleaning in 66.7%. Microscopy provided no additional benefits in terms of exposure in any case, and provided added benefit for cleaning in only 21.4% of cases.For outpatient postoperative care of open mastoidectomy cavities, ear endoscopy provides greater benefit over ear microscopy than vice-versa. In over half of all cases, endoscopy was able to expose areas not visualized under the microscope. Furthermore, in two-thirds of cases, endoscopy enabled removal of material that could not be cleared under microscopy. Ear endoscopy was superior to microscopy in terms of enabling exposure and cleaning of hard-to-reach sites, due to its wider field of vision.Ear endoscopy is a feasible technique for the postoperative management of open mastoidectomy cavities. Ear endoscopy provided superior advantages in terms of exposure and aural cleaning compared with microscopy. Since the very first reports of endoscopic examination of the ear in 1967 –5. The rDespite greatly increased interest in otoendoscopic surgery in recent years, there is a lack of literature on the outpatient use of ear endoscopy for postoperative care. Whether its potential benefits are real has yet to be determined. The importance of postoperative follow-up after otologic surgery is widely known, especially after canal wall down mastoidectomy performed for cholesteatoma. On average, most patients with open mastoidectomy cavities will require 12 visits for exploration and cleaning every 5 years . DespiteThe wide field of view of endoscopes enables a more comprehensive examination of the mastoid cavity, even when considering narrowing of the ear canal . FurtherDespite the benefits described, additional assessment of ear endoscopy is still necessary to determine its actual role in postoperative follow-up. In this setting, endoscopy could represent an alternative tool for patient assessment or even change the way in which follow-up is conducted. Moreover, routine use in follow-up would be a good way of training otolaryngologists in the endoscopic management of the ear.The present study sought to assess the use of ear endoscopy in the postoperative management of open mastoidectomy cavities and to test whether ear endoscopy improves inspection and cleaning capabilities compared with ear microscopy.This prospective study was conducted at the outpatient ENT clinic of a tertiary teaching hospital. Patients who were receiving periodic follow-up at the clinic were invited to take part in the study and provided written informed consent for participation. Data collection took place from January to July 2014. This study was approved by the Universidade de Brasília School of Medicine Research Ethics Committee.The inclusion criteria were: age 18–70 years; history of canal wall down tympanomastoidectomy performed at least 60 days before enrollment; and presence of crust/debris/discharge in the mastoid cavity and/or middle ear requiring removal. The exclusion criterion was presence of surgical complications. In patients who underwent bilateral operation, only the ear that was operated first was included in the sample.Patients were divided into two groups. Over 3 consecutive months, all patients who presented spontaneously to the clinic for visits, were eligible, and agreed to take part in the study were allocated into group 1. The same procedure was then followed for group 2.Ear microscopy was performed with patients in the lying position, using a light microscope (f = 250 mm). Ear endoscopy was performed with patients in the seated position using a 4-mm, 165-mm, 0° Karl Storz™ scope, endoscopy set with camera and 250W halogen light source. Forceps and suction tips were those used routinely for ear microscopy procedures.In group 1, patients received standard care, which consisted of microscopic exploration and cleaning performed by an examiner (an otology fellow) blinded to the study objectives. Each patient was then assessed by a second examiner (a rhinology fellow), who performed endoscopic exploration. If there was any remaining material in the cavity, additional cleaning was performed under endoscopic visualization. The entire endoscopy procedure was recorded. Both fellows were recently graduated ENT specialists who worked in a subspecialty area of the study hospital as part of their respective fellowship programs. Thus, both had similar skills and experience.The second examiner completed Questionnaire 2, which was designed primarily to assess exposure, presence of remaining material in the cavity, and cleaning ability  followed by microscopic examination (performed by the otology fellow), using the same questionnaires.For analysis of results (exposure and cleaning), we considered the benefits provided by the second method in relation to the first. Therefore, in group 1, we assessed whether endoscopy was able to improve exposure or cleaning after the microscope approach. For instance, when complete exposure and cleaning were achieved under microscopic visualization followed by complete exposure and cleaning under endoscopic visualization, we concluded that the second method provided no benefit in relation to the first. When exposure was considered incomplete under microscopy and complete with endoscopy, we regarded the second method as beneficial in relation to the first. When the first examiner graded cleaning as complete but the second examiner found remnants of material requiring removal, we concluded that the second method provided added benefit in terms of both exposure and cleaning  on the results.All variables of interest were categorical. We tested whether each variable  impacted the primary outcomes (benefit for exposure and benefit for cleaning). We also tested for differences between groups in each variable, as well as for independence between two crossed variables, using Fisher’s exact and chi-squared tests. All tests were performed in SPSS 20.0.The study sample comprised 32 patients. Age ranged between 18–70 years . Group 1 comprised 18 patients , whereas group 2 comprised 14 ears . Overall, there were 12 right and 6 left ears in group 1 and 8 right and 6 left ears in group 2. There were no significant between-group differences in these variables. No patient was excluded from the study.p = 0.1812; amount of material, p = 0.2071; technical difficulty, p = 0.1289).Examiner findings regarding meatoplasty size, amount of material requiring removal, and technical difficulty are available as supplemental data . Again, p < 0.0002).In group 1 (microscopy before endoscopy), endoscopic examination provided exposure benefits in 61.1% of cases (11/18). Therefore, in over half of patients, the endoscope was able to expose areas not visualized under microscopy. In group 2 (endoscopy before microscopy), subsequent microscopic examination did not provide exposure benefits in any case (0/14). Comparison of results between the groups  showed aAlso in group 1, endoscopy provided benefits for cavity cleaning in 66.7% of cases (12/18); again, in half of cases, endoscopy enabled removal of material not cleared under microscopic visualization. This figure refers both to cases in which the material was not visualized under the microscope and those in which the examiner detected material but was technically unable to remove it.p = 0.013). In other words, endoscopy provided more benefit over microscopy than vice-versa . Comparison between groups showed a significant difference . Greater difficulty in cleaning was associated with less exposure benefit and cleaning benefit .The results of this study demonstrate that, for outpatient postoperative care of open mastoidectomy cavities, ear endoscopy provides greater benefit over ear microscopy than vice-versa. In over half of all cases (61.1%), endoscopy was able to expose areas not visualized under the microscope. Furthermore, in two-thirds of cases (66.7%), endoscopy enabled removal of material that could not be cleared under microscopy. Endoscopic exploration and cleaning of open mastoidectomy cavities was thus more effective and efficient than the current standard of care (microscopy). Ear endoscopy could replace microscopy without detriment in the majority of situations. Furthermore, ear endoscopy was superior to microscopy in terms of enabling exposure and cleaning of hard-to-reach sites, due to its wider field of vision.Endoscopy allows the examiner to transfer the source of light into the cavity, despite the small size of the ear canal. When combined with the wide field of vision, this enables broader, global visualization, including of otherwise inaccessible recesses and corners. Conversely, microscopy uses a light source projected from outside the canal. Therefore, illumination is greatly limited by the external opening of the ear canal and by the meatoplasty. Contrary to what one might speculate, the limitation of having only one hand free to manipulate the cavity under endoscopy did not cause any significant impairment. The wider view of the cavity enabled adequate manipulation of the material even with one hand only.We did not assess whether the advantages provided by each method were associated with any clinical benefit . The purpose of this study was to assess the applicability of ear endoscopy to procedures already performed routinely under microscopic visualization. The persistence of debris in the mastoid cavity can facilitate persistence of infection. In these cases, the better removal of debris provided by ear endoscopy could contribute to better clinical control of these patients.The fact that the first examiner was aware that the second examiner would conduct a new assessment (despite having no knowledge of the study design or purpose) could be a source of bias. The first examiner might, for instance, be less meticulous in his cleaning due to the knowledge that a second procedure would be performed. Instead, this caused a Hawthorne effect: as the first examiner knew his work would be assessed by the second, he will have tended to perform a more rigorous cleanout, thus minimizing potential bias.The results of ear endoscopy could have been even superior if better equipment had been used. The endoscope itself was a standard nasal examination scope , a model widely available at ENT clinics. Forceps and suction tips were those used routinely for ear microscopy. In cases in which not all debris or discharge could be removed under endoscopy, difficulty manipulating the forceps within the same space occupied by the scope was often the cause. This could be minimized by using narrower wide-field endoscopes, as is already the practice of some otologists, and forceps designed specifically for endoscopy. This would provide more space for manipulation without detriment to the field of view.One limitation of this study is that, although both fellows were recently graduated ENT specialists with similar skills and experience, the second group was assessed by examiners with 3 months of additional experience. Future studies could avoid this limitation by using an examiner cross-over design and involving a greater number of fellows.There is a lack of studies on the outpatient use of ear endoscopy for postoperative clinical care. Despite the growth of ear endoscopy in recent decades –5, its rThis is the first study to assess the outpatient application of otoendoscopy in clinical care of patients with open mastoidectomy cavities. Ear endoscopy requires training–an aspect to be tackled by ear surgeons. Its routine use for outpatient postoperative management could be a way of ensuring continuous training, thus helping with the learning curve and providing benefits for surgeons and patients alike.We conclude that ear endoscopy is an effective tool for postoperative care of open mastoidectomy cavities. In this study, ear endoscopy provided superior exposure and cleaning compared with microscopy. Ear endoscopy might thus be used for these procedures with results at least similar to those obtained with microscopy, and should be used as a supplemental technique whenever feasible.S1 Appendix(TIF)Click here for additional data file.S2 Appendix(TIF)Click here for additional data file.S3 Appendix(TIF)Click here for additional data file.


Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of β-catenin protein, especially S33/S37/T41 phospho-β-catenin, were markedly increased in Usp9xY−/ embryonic cortices. Loss of Usp9x altered composition of the β-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-β-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9xY−/ embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors.Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants.  Post-translational protein modifications play critical roles in mediating and dynamically integrating signalling pathways. Although the role of phosphorylation in signal transduction is well established it is clear that ubiquitylation/deubiquitylation also play critical roles in both NP fate and signal pathway regulation2. In the current study we focused on the role of deubiquitylating enzyme (DUB), ubiquitin specific peptidase 9 located in the X-chromosome (Usp9x) in the regulation of NP function in the mouse neocortex.Neural progenitors (NPs) are the founding cell population driving brain development. Therefore, cell fate decisions taken by NPs must balance their differentiation into neuronal and glial lineages, with self-renewal thereby facilitating ongoing growth. The fate of a NP is temporally and spatially controlled by the simultaneous exposure to multiple extrinsic signalling pathways to which NPs need to coordinate appropriate intrinsic responsesUsp9x is highly expressed in both embryonic and adult NPs5. Conditional deletion of Usp9x from mouse NPs results in perinatal lethality and disrupts the cytoarchitecture of the ventricular and sub ventricular zones in late stage embryos6. Conversely overexpression of Usp9x in mouse embryonic stem cell-derived NPs significantly increased their polarity and self-renewal without affecting their differentiation capacity3. Although these studies demonstrate the importance of Usp9x in NP fate specification, the underlying molecular mechanism(s) remains to be elucidated. To date more than 35 proteins have been reported as Usp9x substrates, many of which are components of intrinsic and extrinsic signalling pathways known to regulate NP function5. However, as these Usp9x-substrate interactions were elucidated in diverse biological systems, and are often cell context specific, further characterisation of Usp9x’s role in NPs is required.Usp9x is ideally situated to integrate extrinsic signals with intrinsic responses in NPs. 1 and Usp9x regulates both in a number of polarised cells. Adherens junctions (AJ) are the predominant cell-cell adhesive structures in NPs and, Usp9x substrates AF-67 and EFA68 are key functional adhesion components. In addition Usp9x has been proposed to promote the trafficking of cadherin-catenin heterodimers during de novo AJ formation of T84 epithelial cells9. Deubiquitylation by Usp9x also activates the polarity regulatory proteins AMP-activated protein kinase related kinesin proteins NUAK1 and MARK4 in polarised epithelia11. Functionally, cellular polarity was up regulated with modest overexpression of Usp9x in embryonic stem cell-derived NPs3.Cell adhesion and apical-basal polarity are key intrinsic regulators of NP fate12. The best-characterised role of Notch signalling in neural development is the maintenance of NP self-renewal. Usp9x is capable of regulating the Notch pathway at multiple levels. Usp9x promotes Notch signalling by antagonising the degradation of Mind bomb1, as well as activating Epsin in the signal sending cell14. Conversely, Usp9x potentially opposes Notch signalling by stabilising the Itch E3 ligase15, which targets the Notch intracellular domain (NICD) for proteasomal degradation in the signal receiving cell. Although no direct evidence has been reported on Usp9x regulation of Wnt signalling, the catalytic domain of Usp9x binds β-catenin and increases the half-life of the cytosolic pool16. Recently, two studies also identified Usp9x associated with the β-catenin destruction complex in HEK293 cells18 suggesting another intersection between Usp9x and Wnt signalling. Therefore Usp9x interacts with both intrinsic and extrinsic mechanisms regulating NP fate specification. In the current study we sought to directly determine if Usp9x regulates NP function in vivo and if so, determine the molecular mechanism(s) involved.Usp9x also regulates components of the Notch and Wnt signalling pathways, which are prominent extrinsic signalling pathways specifying NP fateNestin-cre mediated deletion of Usp9x from mouse NPs results in their disorganisation, and that of their derivatives, within the neocortex of late stage embryos . However, earlier time points have not been investigated. In this study we directly examined the role of Usp9x in NPs using Usp9x conditionally deleted mice (Usp9xY−/) following mating of Usp9xflox/flox females with Nestin-Cre males6.Usp9xY−/ animals compared to litter mate controls (Usp9xY+/) (n = 7) identified E12.5 as the earliest stage at which Usp9x protein was depleted from Usp9xY−/ neocortices  (n = 3)    Fig. . Similar 3) Fig. . Signifi 3) Fig. . Immunor 3) Fig. . Signifi 3) Fig. . TogetheNPs Fig.  suggestiUsp9xY−/ E12.5 and E14.5 mouse neocortical tissues. At E12.5 over 1000 genes were differentially expressed between the Usp9xY+/ and Usp9xY−/ samples, at a significance level of p < 0.05 (n = 4 for each genotype) . Although no difference was observed in the number of PH3-positive (PH3+) mitotic cells at the ventricular surface between Usp9xY+/ and Usp9xY−/ neocortices at either E12.5 or E14.5, a 6-fold increase in abventricular PH3+ nuclei was detected at E12.5     but not E14.5   but not at E14.5   Fig. , and a 2 4) Fig.  in Usp9x 3) Fig. . An incr 3) Fig.  was alsoive Fig. , consist 3) Fig. . At E12.les Fig. .Figure 2+ NPs in either E12.5 or E14.5 Usp9xY−/ brains (n = 3)  neuroblast between Usp9xY+/ and Usp9xY−/ brains in any embryonic stage  (n = 3) . To ensure this was not due to the loss of early born deep-layered neurons in Usp9xY−/ brains, apoptosis was examined using cleaved Caspase3 (Cas3) antibodies and TUNEL assay at E12.5, E14.5, E16.5 and E18.5. No difference was observed in Usp9xY−/ brains with either Cas3  Fig. . Similar 3) Fig.  suggesti. + Fig.  or Ctip2. + Fig.  neurons Usp9xY−/ brains in both IHC and IB analyses, across all embryonic stages examined (n = 3)   Fig. . Even at 3) Fig. . Therefo25. Under normal physiological conditions there are three cellular pools of β-catenin in NPs; (i) membrane associated β-catenin at AJs, (ii) soluble cytoplasmic β-catenin and, (iii) transcriptionally activated β-catenin in the nucleus24. Therefore β-catenin levels in each cellular pool were measured to identify which β-catenin species was upregulated in Usp9xY−/ NPs.β-catenin acts as both an essential linker protein in cadherin-based cell adhesion as well as a co-activator of canonical Wnt signallingUsp9xY−/ brains suggesting the increased β-catenin protein levels were not attributable to its accumulation at AJs. The cytoplasmic and nuclear pools of β-catenin were also assessed. Under physiological conditions the majority of cytoplasmic β-catenin is efficiently bound, phosphorylated and ubiquitylated by the β-catenin destruction complex17. This sub-population of β-catenin can be identified by the phosphorylation of the amino acid residues Serine 33/37 and Threonine 41 (pβ-catenin33/37/41). Strikingly, a 6-fold increase in pβ-catenin33/37/41 was detected in Usp9xY−/ brains at E14.5 (n = 3)   Fig. . In addi 3) Fig. .in-vitro systems27. In line with these studies, Usp9x was co-immunopercipitated with β-catenin from E14.5 Usp9xY+/ neocortical lysates extending this interaction in-vivo    Fig. . Ubiquittes Fig.  implyingtes Fig. .Usp9xY−/ brains activated the Wnt signalling pathway by assaying target gene levels using qRT-PCR. Consistent with the preceding results significantly increased Ccnd1 and Axin2 gene expression levels were observed in the Usp9xY−/ brains at E12.5, E14.5 and E16.5 (n = 3) . Ingenuity pathway analysis conducted on the microarray data independently identified Wnt signalling as one the most significantly affected pathways in Usp9xY−/ brains  Fig. . Howeverns Table . FurtherTherefore, to test if loss of Usp9x can directly activate Wnt signalling, TCF-TOPflash reporter assays were performed on HEK293 cells from which USP9X was depleted with siRNA. USP9X protein level was depleted 72 hours after siRNA transfection and a clear increase in pβ-catenin33/37/41 protein level was observed  but significant increase in AXIN2 expression (n = 3)   Fig. . To detent) Fig. . A caveat3a Fig.  in ReNce40. However, no overt changes in either neurogenesis or total NP numbers were observed in early  Usp9xY−/ brains. One possible explanation for this apparent discrepancy could reside with Usp9x’s regulation of other signalling pathways including Notch, which opposes Wnt signalling in multiple progenitor populations43. Usp9x regulates components of the Notch pathway in both signal sending and receiving cells44. Therefore, we hypothesised that Notch signalling might also be altered in Usp9xY−/ NPs.Over-expression of stabilised forms of β-catenin and activation of Wnt signalling induce both NP self-renewal and neurogenesis during early and mid-neurogenic periods, respectivelyrly Fig.  or late Usp9xY−/ brains and a 50% increase was detected at both E12.5 and E14.5 (p < 0.05)   expression, a NP-specific Notch target47 was examined in the Usp9xY−/ NPs. Consistent with preceding results increased BLBP protein expression was observed in E12.5, E14.5 and E16.5 Usp9xY−/ neocortical tissue (n = 3)   Fig.  raising 05) Fig. . Much sm6.5 Fig. . However 3) Fig. . Increas 3) Fig.  or E18.5 3) Fig. , respectUsp9xY−/ brains could result from increased receptor activation and/or impaired NICD degradation. Mind bomb1 is an Usp9x substrate, which promotes Notch signalling by activating ligands48 in turn leading to Notch receptor activation and increased NICD levels. However, no differences were observed in Mind bomb1 expression in E12.5 or E14.5 Usp9xY−/ brains (n = 3)    Fig.  implying 3) Fig. . Interesins Fig. . These d51 and LNX52 targeting it for proteasomal degradation. However, no DUB has been reported to interact with NUMB. Therefore we sought to determine whether Usp9x binds Numb in the embryonic neocortex. Co-immunoprecipitation with Usp9x antibody from E14.5 Usp9xY+/ cortical lysates detected a strong interaction with Numb and, to a lesser extent, Itch tch Fig. . Reciprotch Fig. . In additch Fig. . These r21. Usp9x is required for normal brain development6 but it was not clear what role, if any, it played in NPs. Nestin-Cre expression resulted in the complete deletion of Usp9x protein from NPs by E12.5 and expression of both AJ and polarity proteins was significantly decreased in Usp9xY−/ NPs at this stage  that the components required to progressively phosphorylate β-catenin are present and functional in the destruction complex and, (ii) that either the progression of phosphorylated β-catenin or the activity of the ubiquitylation machinery within the destruction complex is hindered by the absence of Usp9x. Immunoblot analysis revealed the cellular levels of destruction complex proteins were unaltered and, immunoprecipitation confirmed the endogenous proteins interacted in the absence of Usp9x  the differing roles of Usp9x in L-cells and embryonic NPs and/or (ii) the overexpression of Usp9x’s catalytic domain alone raises concerns about substrate specificity. USP9X also interacts with the cadherin-catenin homodimer at points of protein trafficking in semi-confluent T84 epithelial cells implying a subpopulation of USP9X interacts with the AJ-bound β-catenin . The co-immunoprecipitation of Usp9x with N-cadherin and β-catenin in the embryonic neocortex suggests this interaction also occurs in NPs in vivo. However this pool of β-catenin is not affected following Usp9x deletion and so did not alter total β-catenin levels.β-catenin protein levels are regulated by several DUBs including Usp7, Usp34, Usp4 and Usp47Usp9xY−/ neocortices. However, the data indicate that any effect of Usp9x depletion on Wnt signalling is complex and cell context specific. While pβ-catenin33/37/41 levels were increased 6-fold in Usp9xY−/ neocortices, the transcriptionally active form Tyr654 pβ-catenin was only slightly elevated  in-vivo but expression of Axin2 was only marginally elevated (3%)  and AXIN2 (25%) expression was detected in the ReNcell VM human NP cell line  Fig. . A similine Fig. . Inductit3a Fig. . Use of t3a Fig. . FurtherCcnd1 is not an exclusive Wnt target gene but is regulated by numerous signalling pathways, including Notch61.However, while ingenuity pathway analysis identified the Wnt pathway as significantly affected, the detected transcripts were components of Wnt pathway rather than targets. In addition, Usp9xY−/ brains. The depletion of the E3 ubiquitin ligase Itch was not unexpected as Usp9x recuses it from auto-ubiquitylation and proteasomal degradation in multiple cell types15. However, the observation that Usp9x co-immunoprecipitates and co-localizes  were generated by mating Usp9xloxP/loxP female mice with Nestin-Cre male mice and the genotyping was conducted as described previously6.All animals were used and maintained in accordance with guidelines approved by the Griffith University Animal Ethics Committee under the animal ethics application BPS 02/11 and ESK06/14. The study followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Usp9x conditionally deleted mice  and maintained according to the manufactures instructions. Doxycycline-inducible Usp9x knock-down ReNcell VMs were generated as described previously68.HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum at 37 °C in an incubator with 5% COTime-mated mice were sacrificed by cervical dislocation; embryos were removed and dissected in ice-cold HBSS. Extracted embryonic heads were dropped fixed in 4% paraformaldehyde in PBS. The heads were then cryoprotected by immersion in 15% sucrose and 30% sucrose gradients and finally embedded in Tissue-Tek Optimal Cutting Temperature compound (OTC) (Sakura). 10 μm coronal sections were made using Leica cryostat and mounted on Super Frost Plus microscope slides (Thermo Fisher Scientific). The sections were permeabilized using 1% SDS for 4 minutes then blocked with 2% BSA for 30 minutes at room temperature. Primary and secondary antibodies diluted in 2% BSA were incubated overnight at 4 °C and 1h at RT respectively. The used antibodies and their dilutions were listed in the Table 69. Briefly, the VZ thickness was measured in a confocal image stained with SOX2 by first drawing a line at right angles to the ventricular surface, from the apical surface to the pial surface. The length of this line was measured by ImageJ 1.60s software  and considered total cortical thickness. A line drawn from the apical to basal side of the SOX2-positive band was considered as VZ thickness. The average NP population was calculated as a ratio between the VZ and cortex thicknesses at five different points along the rostral-caudal axis in each image and the mean value of this ratio was used for all statistical analysis. The same method was used for the DCX positive cortical plate to measure the average neuronal population.The cortex was defined as the area from the cortical hem to the pallial-subpallial boundary in each confocal image. Average NP and neuronal populations were measured as described6. Antibodies used are listed in Table Forebrains were dissected from extracted embryos, flash frozen with liquid Nitrogen and stored in -80 until use. Cortical tissues were lysed using lysis buffer  with 100 μl/mL protease and phosphatase inhibitor mix . Protein lysate was processed for immunoblotting and visualised with ECL substrate as describedProteins were extracted from embryonic mouse cortical tissues by sonication in the presence of IP lysis buffer - 20 mM HEPES pH 7.8, 400 mM KCl, 5 mM EDTA, 0.4% NP40, 10% glycerol, 1 mM DTT, protease and phosphatase inhibitor cocktails . Immunoprecipitation was conducted using Pierce co-immunoprecipitation kit (Thermo Scientific) according to manufacturer’s instructions before being subjected to immunoblot analysis.70. Differentially expressed transcripts were identified using the limma package - . Microarray data was deposited into ArrayExpress (Accession Number E-MTAB-4150). Differentially expressed genes were clustered using Euclidean distance65 in Cluster 3.0 and visualized in Java Freeview66.RNA was isolated from cells or embryonic tissue using Trizol reagent (Invitrogen). The cDNA was generated using 2 μg of isolated RNA using the High capacity Reverse transcription kit (Applied Biosystem) according to the manufacturer’s instructions. Quantitative real reactions were conducted on the Rotor gene 6000 (Corbett Life Science) using the SensiFAST HRM kit (Bioline). All samples were run in quadruples and normalised to GAPDH expression. Primer sequences listed in Table 72 using Lipofectamine 2000 reagent (Invitrogen). Wnt3A or DKK1 added to the cells 24 hours after the transfection. 1 hour after the Wnt3a (100 ng/µl) (R&D system Cat# 5036-WN) and DKK1 (100 ng/µl) (Sigma Cat# SRO3258) treatment, cells were lysed using luciferase lysis buffer (Promega). To measure the luciferase activity, 10 μl of cell lysate was transferred to a black 96-well Optiplate (Perkin Elmer). Luciferase activities were performed in triplicate.USP9X expression was depleted in HEK293 cells using USP9X siRNA obtained from Millenium Science (SMARTpool ON-TARGETplus USP9x siRNA Cat# L-006099-00-0005) was used to knockdown USP9X. Scrambled siRNA (ON-TARGETplus Non-targeting Pool Cat# D-001810-10-05) was used as control. USP9X-depleted HEK293 cells were then transfected with plasmids pGL3-TOP and pGL3-FOP containing TCF luciferase-reporter constructStatistical analysis was performed using Prism 5 (GraphPad Prism). For all analysis at least three biological repeats were assessed. Statistical analysis was perform using one-way ANOVA and Tukey’s post-hoc tests, for experiments containing more than two samples, or with a Student T-test for data sets with only two samples. The error bars on all the graphs represent SEM. All the values were presented as mean ± SEM.Supplementary FiguresSupplementary Tables