Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

PARP inhibitor exerts an anti-tumor effect via LMO2 and synergizes with cisplatin in natural killer/T cell lymphoma.

BACKGROUND: PARP inhibitor (PARPi), as a kind of DNA damage repair inhibitor, has been shown to be effective in various solid tumors and hematologic malignancies. Natural killer/T cell lymphoma (NKTCL) is a highly aggressive malignancy, the treatment of which has long been a major challenge in the clinic. Here, we investigated the efficacy and mechanism of PARPi, and the therapeutic value of PARPi combined with cisplatin in NKTCL. METHODS: The cell proliferation, cell apoptosis, and cell cycle of NKTCL cells were detected respectively by CCK-8 and flow cytometry. The changes of mRNA expression and protein level were measured respectively by mRNA-sequencing, quantitative real-time PCR, western blotting, and immunofluorescence. LMO2 expression was detected by immunohistochemistry and western blotting. Targeted knockdown of LMO2 was conducted by short hairpin RNA. The tumor xenograft models were established to evaluate the efficacy of drugs in vivo. RESULTS: PARPi inhibited cell proliferation, promoted cell apoptosis, and induced S-phase cell cycle arrest in NKTCL cells. PARPi led to the accumulation of DNA damage by blocking DNA repair and DNA replication. Additionally, LMO2 deficiency reduced the sensitivity of NKTCL cells to PARPi. Finally, the combination of PARPi and cisplatin exhibited significant synergistic effects both in vitro and in vivo. CONCLUSIONS: In summary, we found that PARPi exerted an anti-tumor effect via LMO2 and synergized with cisplatin in NKTCL, which provides the theoretical basis for the clinical application of PARPi.

AdipoAI suppresses osteoclastogenesis by activating AdipoR1/APPL1: An in vivo experimental study in diabetes-associated peri-implantitis.

AIM: Diabetics experience severe peri-implant inflammatory bone damage. We aimed to provide powerful evidence supporting the novel adiponectin receptor agonist AdipoAI in treating diabetes-associated peri-implantitis. MATERIALS AND METHODS: Twenty-four ZDF-Leprfa/Crl rats were randomly allocated to three groups (N = 8). After feeding with a high-fat diet to establish diabetic rats, experimental peri-implantitis was induced by implanting titanium rods (1.5 mm diameter and 20 mm length) contaminated with Staphylococcus aureus into the femurs. Radiographic evaluation, microCT, histological analyses and qRT-PCR were used to detect inflammatory infiltration and bone destruction. In vitro, the inhibition by AdipoAI of osteoclastogenesis, including the number and function of osteoclasts, was investigated by TRAP staining, immunofluorescence, qRT-PCR and Western blotting. Immunofluorescence, qRT-PCR and Western blotting were also utilized to explore AdipoR1, APPL1, NF-κB and Wnt5a-Ror2 signalling molecules in this process. One-way ANOVA with Tukey's post hoc test was used to compare the data. RESULTS: AdipoAI reduced inflammation and bone destruction caused by peri-implantitis in diabetic rats, which were manifested by a reduction in F4/80-positive macrophage infiltration by 72%, the number of osteoclasts by 58% and the levels of cytokines (p < .05) in disease group. In vitro, 1 µM AdipoAI decreased the number of osteoclasts to 51%, inhibited F-actin ring formation and reduced the levels of related markers (p < .05). Mechanistically, AdipoAI activated AdipoR1/APPL1 and conversely suppressed the phosphorylation of IκB-α, nuclear translocation of P65 and the Wnt5a-Ror2 signalling pathway (p < .05). CONCLUSIONS: AdipoAI suppressed osteoclastogenesis in diabetes-associated peri-implantitis by inhibiting the NF-κB and Wnt5a-Ror2 pathways via the AdipoR1/APPL1 axis.

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic ß-cells.

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in ß-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic ß-cells. ß-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic ß-cells.

Targeting MAGI2-AS3-modulated Akt-dependent ATP-binding cassette transporters as a possible strategy to reverse temozolomide resistance in temozolomide-resistant glioblastoma cells.

Drug resistance is a major impediment to the successful treatment of glioma. This study aimed to elucidate the effects and mechanisms of the long noncoding RNA membrane-associated guanylate kinase inverted-2 antisense RNA 3 (MAGI2-AS3) on temozolomide (TMZ) resistance in glioma cells. MAGI2-AS3 expression in TMZ-resistant glioblastoma (GBM) cells was analyzed using the Gene Expression Omnibus data set GSE113510 and quantitative real-time PCR (qRT-PCR). Cell viability and TMZ half-maximal inhibitory concentration values were determined using the MTT assay. Apoptosis and cell cycle distribution were evaluated using flow cytometry. The expression of multidrug resistance 1 (MDR1), ATP-binding cassette superfamily G member 2 (ABCG2), protein kinase B (Akt), and phosphorylated Akt was detected using qRT-PCR and/or western blot analysis. MAGI2-AS3 was expressed at low levels in TMZ-resistant GBM cells relative to that in their parental cells. MAGI2-AS3 re-expression alleviated TMZ resistance in TMZ-resistant GBM cells. MAGI2-AS3 overexpression also accelerated TMZ-induced apoptosis and G2/M phase arrest. Mechanistically, MAGI2-AS3 overexpression reduced MDR1 and ABCG2 expression and inhibited the Akt pathway, whereas Akt overexpression abrogated the reduction in MDR1 and ABCG2 expression induced by MAGI2-AS3. Moreover, activation of the Akt pathway inhibited the effects of MAGI2-AS3 on TMZ resistance. MAGI2-AS3 inhibited tumor growth and enhanced the suppressive effect of TMZ on glioma tumorigenesis in vivo. In conclusion, MAGI2-AS3 reverses TMZ resistance in glioma cells by inactivating the Akt pathway.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration.

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.

BAG3 induces α-SMA expression in human fibroblasts and its over-expression correlates with poorer survival in fibrotic cancer patients.

Hypoxia and angiogenesis in solid tumors are often strictly linked to the development of fibrotic tissues, a detrimental event that compromises the antitumor immunity. As a consequence, tumor aggressiveness and poor patient prognosis relate to higher incidence of tissue fibrosis and stromal stiffness. The molecular pathways through which normal fibroblasts are converted in cancer-associated fibroblasts (CAFs) have a central role in the onset of fibrosis in tumor stroma, thus emerging as a strategic target of novel therapeutic approaches for cancer disease. Several studies addressed the role of BAG3 in sustaining growth and survival of cancer cell and also shed light on the different mechanisms in which the intracellular protein is involved. More recently, new pieces of evidence revealed a pivotal role of extracellular BAG3 in pro-tumor cell signaling in the tumor microenvironment, as well as its involvement in the development of fibrosis in tumor tissues. Here we report further data showing the presence of the BAG3 receptor (Interferon-induced transmembrane protein [IFITM]-2) on the plasma membrane of normal dermal fibroblasts and the activity of BAG3 as a factor able to induce the expression of α-smooth muscle actin and the phosphorylation of AKT and focal adhesion kinase, that sustain CAF functions in tumor microenvironment. Furthermore, in agreement with these findings, bag3 gene expression has been analyzed by high throughput RNA sequencing databases from patients-derived xenografts. A strong correlation between bag3 gene expression and patients' survival was found in several types of fibrotic tumors. The results obtained provide encouraging data that identify BAG3 as a promising therapeutic target to counteract fibrosis in tumors.

CircASAP1 promotes the development of cervical cancer through sponging miR-338-3p to upregulate RPP25.

Circular RNAs have been identified as vital regulators to regulate the development of human cancers, including cervical cancer. Therefore, this study was designed to clarify the underlying mechanism of circASAP1 in cervical cancer. The real-time quantitative PCR assay was applied to quantify the expression levels of circASAP1, microRNA (miR)-338-3p, and ribonuclease P and MRP subunit p25 (RPP25) in cervical cancer tissues and cells. The cell proliferation ability was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide and colony-forming assays. The protein expression levels of cyclin D1, proliferating cell nuclear antigen, and RPP25 were assessed by western blot assay. Flow cytometry assays were used to determine the apoptosis and cell cycle distribution of cervical cancer cells. The transwell assay was employed to test the migration and invasion abilities of cervical cancer cells. The interaction relationship between miR-338-3p and circASAP1 or RPP25 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. The xenograft experiment was established to clarify the functional role of circASAP1 inhibition in vivo. CircASAP1 was overexpressed in cervical cancer tissues and cells compared with negative groups. Additionally, the loss-of-functional experiments implied that knockdown of circASAP1 impeded proliferation, migration, and invasion while induced apoptosis and cell cycle arrest in cervical cancer cells along with repressed tumor growth in vivo through regulation of miR-338-3p. In addition, RPP25 was a target mRNA of miR-338-3p, and overexpression of miR-338-3p suppressed proliferation, migration, and invasion while induced apoptosis and cell cycle arrest in cervical cancer cells by suppressing RPP25 expression. Mechanistically, circASAP1 could function as a sponge for miR-338-3p to increase the expression of RPP25, and further regulated proliferation, migration, invasion, apoptosis, and cell cycle program of cervical cancer cells, which might be potential markers for cervical cancer diagnosis.

Network Pharmacology Research and Dual-omic Analyses Reveal the Molecular Mechanism of Natural Product Nodosin Inhibiting Muscle-Invasive Bladder Cancer in Vitro and in Vivo.

Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo, nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.

Targeting TRAF3IP2 alleviates high glucose-induced cardiomyocyte inflammation and apoptosis.

To clarify the role of TRAF3IP2 in high glucose (HG)-stimulated cardiomyocyte inflammation and apoptosis and its action mechanism. SiRNA plasmid of TRAF3IP2 was constructed and transfected into HG-stimulated cardiomyocytes to silence TRAF3IP2. The expression of TRAF3IP2 was determined by quantitative polymerase chain reaction (qPCR) and western blot. Cell viability and cytotoxicity were first observed using cell counting kit-8 and lactate dehydrogenase assays. The inflammatory injury of the cardiomyocytes was then examined by real time-qPCR (RT-qPCR) and western blot. The oxidative stress of the cardiomyocytes was evaluated using reactive oxygen species assay kit, RT-qPCR, western blot and enzyme activity assay kit. Next, cell apoptosis was detected employing TUNEL and western blot. Finally, RT-qPCR and western blot were performed to investigate the effects of inhibitors of dipeptidyl peptidase-4, including saxagliptin, empagliflozin and linagliptin, on TRAF3IP2. TRAF3IP2 expression was found to be increased in HG-stimulated cardiomyocytes. TRAF3IP2 interference inhibited HG-induced cell viability loss, cytotoxicity, inflammatory response, oxidative stress and apoptosis of the cardiomyocytes. Moreover, saxagliptin, empagliflozin and linagliptin inhibited the expression of TRAF3IP2. TRAF3IP2 interference alleviates HG-induced inflammation and apoptosis of cardiomyocytes. The result suggests that TRAF3IP2 may be a promising therapeutic target in treating diabetic cardiomyopathy.

Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer's Disease Mouse Model Carrying a PSEN1 p.G378E Mutation.

Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/-) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1ß and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/- mice) compared with PSEN1G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B.

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Bri2 BRICHOS chaperone rescues impaired fast-spiking interneuron behavior and neuronal network dynamics in an AD mouse model in vitro.

Synchronized and properly balanced electrical activity of neurons is the basis for the brain's ability to process information, to learn, and to remember. In Alzheimer's disease (AD), which causes cognitive decline in patients, this synchronization and balance is disturbed by the accumulation of neuropathological biomarkers such as amyloid-beta peptide (Aß42). Failure of Aß42 clearance mechanisms as well as desynchronization of crucial neuronal classes such as fast-spiking interneurons (FSN) are root causes for the disruption of the cognition-relevant gamma brain rhythm (30-80 Hz) and consequent cognitive impairment observed in AD. Here we show that recombinant BRICHOS molecular chaperone domains from ProSP-C or Bri2, which interfere with Aß42 aggregation, can rescue the gamma rhythm. We demonstrate that Aß42 progressively decreases gamma oscillation power and rhythmicity, disrupts the inhibition/excitation balance in pyramidal cells, and desynchronizes FSN firing during gamma oscillations in the hippocampal CA3 network of mice. Application of the more efficacious Bri2 BRICHOS chaperone rescued the cellular and neuronal network performance from all ongoing Aß42-induced functional impairments. Collectively, our findings offer critical missing data to explain the importance of FSN for normal network function and underscore the therapeutic potential of Bri2 BRICHOS to rescue the disruption of cognition-relevant brain rhythms in AD.

Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models.

Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have not been successful. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing allows to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. We analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition and proliferation, clonogenicity, or spherogenicity as readouts, in human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC). Cultured glioma cells were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. In vivo studies confirmed the anti-glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN-229 glioma xenograft model in nude mice. These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.

Water-soluble variant of human Lynx1 positively modulates synaptic plasticity and ameliorates cognitive impairment associated with α7-nAChR dysfunction.

Lynx1 is a GPI-tethered protein colocalized with nicotinic acetylcholine receptors (nAChRs) in the brain areas important for learning and memory. Previously, we demonstrated that at low micromolar concentrations the water-soluble Lynx1 variant lacking GPI-anchor (ws-Lynx1) acts on α7-nAChRs as a positive allosteric modulator. We hypothesized that ws-Lynx1 could be used for improvement of cognitive processes dependent on nAChRs. Here we showed that 2 µM ws-Lynx1 increased the acetylcholine-evoked current at α7-nAChRs in the rat primary visual cortex L1 interneurons. At higher concentrations ws-Lynx1 inhibits α7-nAChRs expressed in Xenopus laevis oocytes with IC50  ~ 50 µM. In mice, ws-Lynx1 penetrated the blood-brain barrier upon intranasal administration and accumulated in the cortex, hippocampus, and cerebellum. Chronic ws-Lynx1 treatment prevented the olfactory memory and motor learning impairment induced by the α7-nAChRs inhibitor methyllycaconitine (MLA). Enhanced long-term potentiation and increased paired-pulse facilitation ratio were observed in the hippocampal slices incubated with ws-Lynx1 and in the slices from ws-Lynx1-treated mice. Long-term potentiation blockade observed in MLA-treated mice was abolished by ws-Lynx1 co-administration. To understand the mechanism of ws-Lynx1 action, we studied the interaction of ws-Lynx1 and MLA at α7-nAChRs, measured the basal concentrations of endogenous Lynx1 and the α7 nAChR subunit and their association in the mouse brain. Our findings suggest that endogenous Lynx1 limits α7-nAChRs activation in the adult brain. Ws-Lynx1 partially displaces Lynx1 causing positive modulation of α7-nAChRs and enhancement of synaptic plasticity. Ws-Lynx1 and similar compounds may constitute useful hits for treatment of cognitive deficits associated with the cholinergic system dysfunction.

Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.

Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling.

Mitogen-activated protein kinases (MAPK): Erk1 and Erk2 are key players in negative-feedback regulation of fibroblast growth factor (FGF) signaling. Upon activation, Erk1 and Erk2 directly phosphorylate FGF receptor 1 (FGFR1) at a specific serine residue in the C-terminal part of the receptor, substantially reducing the tyrosine phosphorylation in the receptor kinase domain and its signaling. Similarly, active Erks can also phosphorylate multiple threonine residues in the docking protein FGF receptor substrate 2 (FRS2), a major mediator of FGFR signaling. Here, we demonstrate that in NIH3T3 mouse fibroblasts and human osteosarcoma U2OS cells stably expressing FGFR1, in addition to Erk1 and Erk2, p38 kinase is able to phosphorylate FRS2. Simultaneous inhibition of Erk1/2 and p38 kinase led to a significant change in the phosphorylation pattern of FRS2 that in turn resulted in prolonged tyrosine phosphorylation of FGFR1 and FRS2 and in sustained signaling, as compared to the selective inhibition of Erks. Furthermore, excessive activation of p38 with anisomycin partially compensated the lack of Erks activity. These experiments reveal a novel crosstalk between p38 and Erk1/2 in downregulation of FGF-induced signaling.

The common promoter polymorphism rs11666254 downregulates FPR2/ALX expression and increases risk of sepsis in patients with severe trauma.

BACKGROUND: Formyl peptide receptor 2-lipoxin receptor (FPR2/ALX) modulates the anti-inflammatory response and therefore may be a target for treating sepsis. The purpose of this study was to investigate the association between genetic variants of the FPR2/ALX gene and sepsis after severe trauma as well as to further analyze the functions of sepsis-related genetic polymorphisms. METHODS: Three tag single-nucleotide polymorphisms (tag SNPs) that captured all common alleles across the FPR2/ALX genomic region were genotyped using pyrosequencing in an initial sample consisting of 275 patients with severe trauma. The rs11666254 polymorphism, which had statistical significance, was genotyped in an additional 371 patients, and logistic regression analysis was performed to determine associations between the FPR2/ALX gene polymorphism and sepsis susceptibility after severe trauma. The messenger RNA (mRNA) and protein levels of FPR2/ALX in the lipopolysaccharide-stimulated white blood cells of trauma patients were determined by performing quantitative polymerase chain reactions and Western blot analysis. Tumor necrosis factor (TNF)-α production was measured by enzyme-linked immunosorbent assay. The effects of the promoter polymorphism rs11666254 on the transcription activity of FPR2/ALX were analyzed using a luciferase reporter assay. RESULTS: Among the three tag SNPs, only the rs11666254 polymorphism was found to be significantly associated with sepsis in trauma patients, and this association persisted after a pooled analysis of all 646 trauma patients, which showed that patients who carried the A allele of rs11666254 had a significantly higher risk of developing sepsis than individuals who carried the G allele. This SNP was also significantly associated with lower FPR2/ALX mRNA and protein expression as well as higher TNF-α production from the peripheral blood leukocyte response to bacterial lipoprotein stimulation. In addition, the rs11666254 polymorphism could significantly decrease the promoter activity of the FPR2/ALX gene. CONCLUSIONS: The rs11666254 polymorphism in the FPR2/ALX gene is a functional SNP that increases sepsis susceptibility in patients after traumatic injury.