4R Tau Modulates Cocaine-Associated Memory through Adult Dorsal Hippocampal Neurogenesis.

The development, persistence and relapse of drug addiction require drug memory that generally develops with drug administration-paired contextual stimuli. Adult hippocampal neurogenesis (AHN) contributes to cocaine memory formation; however, the underlying mechanism remains unclear. Male mice hippocampal expression of Tau was significantly decreased during the cocaine-associated memory formation. Genetic overexpression of four microtubule-binding repeats Tau (4R Tau) in the mice hippocampus disrupted cocaine memory by suppressing AHN. Furthermore, 4R Tau directly interacted with phosphoinositide 3-kinase (PI3K)-p85 and impaired its nuclear translocation and PI3K-AKT signaling, processes required for hippocampal neuron proliferation. Collectively, 4R Tau modulates cocaine memory formation by disrupting AHN, suggesting a novel mechanism underlying cocaine memory formation and provide a new strategy for the treatment of cocaine addiction.SIGNIFICANCE STATEMENT Drug memory that generally develops with drug-paired contextual stimuli and drug administration is critical for the development, persistence and relapse of drug addiction. Previous studies have suggested that adult hippocampal neurogenesis (AHN) plays a role in cocaine memory formation. Here, we showed that Tau was significantly downregulated in the hippocampus in the cocaine memory formation. Tau knock-out (KO) promoted AHN in the hippocampal dentate gyrus (DG), resulting in the enhanced memory formation evoked by cocaine-cue stimuli. In contrast, genetically overexpressed 4R Tau in the hippocampus disrupted cocaine-cue memory by suppressing AHN. In addition, 4R Tau interacted directly with phosphoinositide 3-kinase (PI3K)-p85 and hindered its nuclear translocation, eventually repressing PI3K-AKT signaling, which is essential for hippocampal neuronal proliferation.

Interaction between CD9 and PI3K­p85 activates the PI3K/AKT signaling pathway in B­lineage acute lymphoblastic leukemia.

Our previous study has shown that CD9 knockdown could suppress cell proliferation, adhesion, migration and invasion, and promote apoptosis and the cytotoxicity of chemotherapeutic drugs in the B­lineage acute lymphoblastic leukemia (B­ALL) cell line SUP­B15. In this study, we further investigated the molecular mechanism underlying the effects of CD9 on leukemic cell progression and the efficacy of chemotherapeutic agents in B­ALL cells. Using the CD9­knockdown SUP­B15 cells, we demonstrated that the silencing of the CD9 gene significantly reduced the expression of phosphorylated­phosphatidylinositol­3 kinase (p­PI3K), phosphorylated­protein kinase B (p­AKT), P­glycoprotein (P­gp), multidrug resistance­associated protein 1 (MRP1), breast cancer resistance protein (BCRP), matrix metalloproteinase 2 (MMP2) and phosphorylated­focal adhesion kinase (p­FAK). In addition, glutathione S­transferase (GST) pull­down assay showed the binding between CD9 and both PI3K­p85α and PI3K­p85ß in vitro, while co­immunoprecipitation assay showed the binding between CD9 and both PI3K­p85α and PI3K­p85ß in vivo. Furthermore, the PI3K/AKT inhibitor LY294002 mirrored the effects of CD9 knockdown in SUP­B15 cells. Taken together, these findings demonstrated that CD9 activates the PI3K/AKT signaling pathway through direct interaction with PI3K­p85 in B­ALL cells. Our data provide evidence for the inhibition of the PI3K/AKT pathway as a novel therapeutic option in CD9 antigen­positive B­ALL.

Evidence for altered insulin signalling in the brains of genetic absence epilepsy rats from Strasbourg.

Insulin-mediated signalling in the brain is critical for neuronal functioning. Insulin resistance is implicated in the development of some neurological diseases, although changes associated with absence epilepsy have not been established yet. Therefore, we examined the major components of PI3K/Akt-mediated insulin signalling in cortical, thalamic, and hippocampal tissues collected from Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic Control (NEC) rats. Insulin levels were also measured in plasma and cerebrospinal fluid (CSF). For the brain samples, the nuclear fraction (NF) and total homogenate (TH) were isolated and investigated for insulin signalling markers including insulin receptor beta (IRß), IR substrate-1 and 2 (IRS1 & 2), phosphatase and tensin homologue (PTEN), phosphoinositide 3-kinase phospho-85 alpha (PI3K p85α), phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate, protein kinase B (PKB/Akt1/2/3), glucose transporter-1 and 4 (GLUT1 & 4) and glycogen synthase kinase-3ß (GSK3ß) using western blotting. A significant increase in PTEN and GSK3ß levels and decreased PI3K p85α and pAkt1/2/3 levels were observed in NF of GAERS cortical and hippocampal tissues. IRß, IRS1, GLUT1, and GLUT4 levels were significantly decreased in hippocampal TH of GAERS compared to NEC. A non-significant increase in insulin levels was observed in plasma and CSF of GAERS rats. An insulin sensitivity assay showed decreased p-Akt level in cortical and hippocampal tissues. Together, altered hippocampal insulin signalling was more prominent in NF and TH compared to cortical and thalamic regions in GAERS. Restoring insulin signalling may improve the pathophysiology displayed by GAERS, including the spike-and-wave discharges that relate to absence seizures in patients.

Inhibitory Effects of Ginsenoside Ro on Clot Retraction through Suppressing PI3K/Akt Signaling Pathway in Human Platelets.

Glycoprotein IIb/IIIa (αIIb/ß3) is the most abundant integrin on platelet surfaces, which is involved in interaction between platelets, and triggers an intracellular signaling cascade, platelet shape changes, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibronectin and fibrinogen to αIIb/ß3 and clot retraction. We found that G-Ro inhibited thrombin-induced platelet aggregation dose-dependently and attenuated the fibronectin-, and fibrinogen-binding to αIIb/ß3 through the dephosphorylation of phosphoinositide 3-kinase p85 and Akt, which influence clot retraction, reflecting the intensification of thrombus. We observed that G-Ro is involved in αIIb/ß3 in human platelets. These results suggest that G-Ro is beneficial, inhibiting fibronectin adhesion, fibrinogen binding, and clot retraction. Therefore, G-Ro in Panax ginseng may prevent platelet aggregation-mediated thrombotic disease.

Interaction between CD133 and PI3K-p85 promotes chemoresistance in gastric cancer cells.

Chemoresistance in gastric cancer is the leading cause of tumor recurrence and poses a substantial therapeutic challenge. The stem cell biomarker CD133 has been implicated in drug resistance of tumor-initiating cells in a number of cancers including gastric cancer. Therefore, we investigated the molecular mechanism of CD133-associated multidrug resistance in gastric cancer cells. Using CD133 overexpressing and knockdown gastric cancer cell lines, we demonstrated that loss of CD133 significantly increased the growth inhibition of chemotherapeutic agents; whereas, overexpression significantly reduced growth inhibition. Furthermore, CD133 knockdown significantly reduced the enzymatic activity of phosphatidylinositol-3 kinase (PI3K) and the expression of P-glycoprotein (P-gp), B-cell lymphoma 2 (BCL2), and phosphorylated-protein kinase B (p-AKT), but elevated the expression of BCL2 associated X (BAX). Conversely, overexpression of CD133 significantly increased PI3K enzymatic activity, expression of P-gp, BCL2, and p-AKT, and decreased BAX expression. The PI3K/AKT inhibitor LY294002 mirrored the effects of loss of CD133; whereas, the PI3K/AKT activator epidermal growth factor reproduced the effects of CD133 overexpression. To identify the interaction between CD133 and PI3K, we used site-directed mutagenesis to mutate individual tyrosine residues of CD133. We found that binding between CD133 and p85, the regulatory subunit of PI3K, was significantly reduced when tyrosine 852 was mutated. In summary, we have demonstrated that CD133 activates the PI3K/AKT signal transduction pathway through direct interaction with PI3K-p85, resulting in multidrug resistance of gastric cancer cells. These results suggest that the interaction between CD133 and PI3K-p85 may offer a novel therapeutic target in multidrug resistant gastric cancer.

Mechanism of soluble beta-amyloid 25-35 neurotoxicity in primary cultured rat cortical neurons.

This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25-35 (Aß25-35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aß25-35 at concentrations of 10nmol/L, 100nmol/L, 1µmol/L, 10µmol/L, or 30µmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aß25-35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aß25-35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aß25-35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways.

[Effects of electroacupuncture on insulin resistance and hypothalamic insulin signal molecule in rats with diet-induced obesity].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on body weight, insulin resistance (IR) and hypothalamic insulin signal molecule in rats with diet-induced obesity (DIO), and to explore the action mechanism of EA on DIO. METHODS: Fifty SD male rats were randomly divided into a low fat diet (LFD) group (10 rats) and a high fat diet (HFD) group (40 rats). Rats were fed with LFD and HFD, respectively, and the DIO model was established in the HFD group. After the model was established, the rats were randomly divided intoa model group, an EA group and a medication group, ten rats in each one. The rats in the EA group were treated with EA at "Housanli" (ST 36) and "Quchi" (LI 11) for 20 min, once a day for totally 28 days. The rats in the medication group were treated with intragastric administration of orlistat, once a day for 28 days. The rats in the LFD group and model group received no treatment. After treatment, HE staining method was applied to observe the morphological changes of liver; the biochemistry technique and radioimmunoassay method were applied to detect the fasting plasma glucose (FPG) and fasting insulin (FINS); Western blot method was applied to measure the expression of phosphatidylinositol-3 kinase p85 subunit (PI3K-p85) and insulin receptor substrate 2 (IRS2). RESULTS: Under light microscope, compared with the model group, the fatty degenerative cells were below 1/2 in the EA group and the medication group, accompanied with decreased lipid droplet, mild edema and none inflammatory infiltration. The body weight, FPG, FINS, homeostasis model assessment-insulin resistance index (HOMA-IR) and PI3K-p85 in the EA group were significantly lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was not significantly different from the model group (P>0.05). The body weight, HOMA-IR and PI3K-p85 in the medication group were lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was higher than that in the model group (P<0.05). The differences of each index were not significant between the EA group and the medication group (all P>0.05). In addition, rats in the medication group showed watery defecation, decreased activity, fatigue mentality and yellow hairs, while rats in the EA group showed normal defecation and vivid hair. CONCLUSIONS: EA can decrease the expression of PI3K-p85 to prompt the IR of DIO rats, inhibit the weight body and improve hepatic steatosis, which is probably one of the action mechanisms of EA on DIO. Besides, the adverse effects as the medication group can be avoided.

MiR-503 targets PI3K p85 and IKK-ß and suppresses progression of non-small cell lung cancer.

A microRNA usually has the ability to coordinately repress multiple target genes and therefore are associated with many pathological conditions such as human cancer. Our understanding of the biological roles of microRNAs in lung cancer, however, remains incomplete. In this study, we identified miR-503 as a tumor-suppressive microRNA in human non-small cell lung carcinoma (NSCLC), whose expression level correlates inversely with overall survival in NSCLC patients. Ectopic expression of miR-503 suppressed tumor cell proliferation and metastasis-related traits in vitro as well as in vivo, supporting a anti-cancer role of the microRNA in NSCLC progression. Mechanistic study revealed that oncogenic PI3K p85 and IKK-ß were direct targets of miR-503. Overexpression of either PI3K p85 or IKK-ß partially restored the malignant properties of NSCLC cells in the presence of miR-503. Taken together, our data demonstrate miR-503 inhibits the malignant phenotype of NSCLC by targeting PI3K p85 and IKK-ß and might play a suppressive role in the pathogenesis of NSCLC, thus providing new insights in developing novel diagnostic and therapeutic approaches.

RNAi targeting AKT1 and PI3K P85 suppresses proliferation of breast carcinoma MCF-7 cells / 中国肿瘤生物治疗杂志

Objective: To investigate the effect of RNA interference (RNAi) targeting AKT1 and PI3K P85 on the proliferation and invasion of breast carcinoma MCF-7 cells. Methods: The recombinant adenovirus expression vector, which contained short hairpin RNA (shRNA) targeting open reading frames of AKT1 and PI3K P85 (rAd5-siAKT1-siPI3K), was transfected into human breast carcinoma MCF-7 cells. AKT1 and PI3K P85 mRNA and protein expressions were detected by real-time PCR and Western blotting analysis. The expressions of PCNA, cyclinD1, and P53 were also detected by Western blotting analysis. The proliferation and apoptosis of MCF-7 cells were measured by MTT, flow cytometry and 2-dementinal and 3-dementional matrigel assay. Results: Recombinant adenovirus vector rAd5-siAKT1-siPI3K dramatically down-regulated AKT1 and PI3K P85 mRNA and protein expressions in MCF-7 cells; the downstream factors PCNA and cyclin D1 were also down-regulated, while P53 was up-regulated. Growth of MCF-7 cells was inhibited by over 50% in rAd5-siAKT1-siPI3K group as measured by MTT assay, and cell cycle was arrested in G_1/G_0 phase compared with untransfected and rAd5-siCtrl transfected groups. Cell growth on matrigel matrix showed normal cell shapes, while the cells in rAd5-siAKT1-siPI3K transfected group were detached from the matrix or grew in scattered clustering patterns, forming only small aggregates. Conclusion: shRNA targeting AKT1 and PI3K P85 can significantly down-regulate the expression of AKT1 and PI3K P85 in breast carcinoma MCF-7 cells, and inhibit the growth of MCF-7 cells in vitro.