Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish.

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that exert crucial functions in regulating gene expression in mammals. However, the function and regulation mechanism of circRNAs in lower vertebrates are still unknown. Here, we discovered a novel circRNA derived from Deltex E3 ubiquitin ligase 1 (Dtx1) gene, namely, circDtx1, which was related to the antiviral responses in teleost fish. Results indicated that circDtx1 played essential roles in host antiviral immunity and inhibition of SCRV replication. Our study also found a microRNA miR-15a-5p, which could inhibit antiviral immune response and promote viral replication by targeting TRIF. Moreover, we also found that the antiviral effect inhibited by miR-15a-5p could be reversed with the circDtx1. In mechanism, our data revealed that circDtx1 was a competing endogenous RNA (ceRNA) of TRIF by sponging miR-15a-5p, leading to activation of the NF-κB/IRF3 pathway, and then enhancing the innate antiviral responses. Our results indicated that circRNAs played a regulatory role in immune responses in teleost fish.

Oxidized low-density lipoprotein activates extracellular signal-regulated kinase signaling to downregulate sortilin expression in liver sinusoidal endothelial cells.

BACKGROUND AND AIM: Both type 2 diabetes mellitus and non-alcoholic fatty liver disease are closely associated with elevated levels of low-density lipoprotein cholesterol and its oxidized form (ox-LDL). This study aimed to investigate the regulation of sortilin in liver tissue and its potential implications for lipid metabolism. METHODS: Sixty male Wistar rats were randomly divided into four groups: control group (n = 15), ox-LDL group (n = 15), PD98059 group (n = 15), and ox-LDL + PD98059 group (n = 15). Liver sinusoidal endothelial cells were extracted from liver tissue of the control group and were identified using an anti-CD31 antibody. Lipid droplet accumulation was observed by Oil red O and hematoxylin-eosin staining. The protein expression levels were detected by immunohistochemical staining, real-time reverse transcription-polymerase chain reaction, and western blot. Histopathologic examinations were performed by Gomori methenamine silver staining. RESULTS: The ox-LDL group exhibited increased lipid droplet accumulation. Further, ox-LDL activated the extracellular signal-regulated kinase (ERK)-mediated downregulation of sortilin expression, whereas blocking of ERK signaling by PD98059 increased sortilin protein expression. Consistently, hematoxylin-eosin staining showed that the structure of the hepatocytes was loose and disordered in arrangement, with lipid droplets present in the cytoplasm of the ox-LDL group. However, PD98059 significantly improved the integration of the scaffold structure. Gomori methenamine silver staining showed that the ox-LDL group had darker and more obvious fragmented silver nitrate deposits in the basement membrane and sinus space. CONCLUSIONS: Sortilin can protect liver sinusoidal endothelial cells from injury and maintain integration of the liver scaffold structure in ox-LDL-induced lipid-injured liver.

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1.

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.

The inflammatory cytokine interferon-gamma inhibits sortilin-1 expression in hepatocytes via the JAK/STAT pathway.

Sortilin-1, a receptor of the VPS10p family, has been associated with cardiovascular disease in genome-wide association studies. It is implicated in lipoprotein metabolism, secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) and secretion of inflammatory cytokines. However, its own regulation remains unclear. Chronic inflammation is a hallmark of atherosclerosis and the absence of regulatory T (Treg) cells is associated with reduced protein expression of sortilin-1 in the liver. Therefore, we postulated that mediator(s) of inflammation known to be downregulated by Treg cells may modulate sortilin-1 expression. In this study, we identify interferon-gamma (IFN-γ) as the key inflammatory mediator controlling sortilin-1 levels. In vitro cultures of murine hepatocytes cell line and in silico experiments showed that the transcription factor Signal transducer and activator of transcription 1 was activated and bound to the Sort-1 gene upon IFN-γ treatment. This reduced the expression of sortilin-1, while disrupting the IFN-γ signaling pathway prevented the effect. These data unravel an intricate mechanism by which inflammation modulates receptors involved in lipoprotein turnover.

Enhanced Proteostasis in Post-ischemic Stroke Mouse Brains by Ubiquilin-1 Promotes Functional Recovery.

Stroke is pathologically associated with oxidative stress, protein damage, and neuronal loss. We previously reported that overexpression of a ubiquitin-like protein, ubiquilin-1 (Ubqln), protects neurons against ischemia-caused brain injury, while knockout of the gene exacerbates cerebral ischemia-caused neuronal damage and delays functional recovery. Although these observations indicate that Ubqln is a potential therapeutic target, transgenic manipulation-caused overexpression of Ubqln occurs before the event of ischemic stroke, and it remains unknown whether delayed Ubqln overexpression in post-ischemic brains within a clinically relevant time frame is still beneficial. To address this question, we generated lentiviruses (LVs) either overexpressing or knocking down mouse Ubqln, and treated post-ischemic stroke mice 6 h following the middle cerebral artery occlusion with the LVs before animal behaviors were evaluated at day 1, 3, 5, and 7. Our data indicate that post-ischemic overexpression of Ubqln significantly promoted functional recovery, whereas post-ischemic downregulation of Ubqln expression delays functional recovery. To further understand the mechanisms underlying how Ubqln functions, we also isolated protein aggregates from the brains of wild-type mice or the mice overexpressing Ubqln following ischemia/reperfusion. Western blot analysis indicates that overexpression of Ubqln significantly reduced the accumulation of protein aggregates. These observations not only suggest that Ubqln is a useful candidate for therapeutic intervention for ischemic stroke but also highlight the significance of proteostasis in functional recovery following stroke.

Stonin 2 Overexpression is Correlated with Unfavorable Prognosis and Tumor Invasion in Epithelial Ovarian Cancer.

Stonin 2 (STON2), which functions in adjusting endocytotic complexes, is probably involved in the monitoring of the internalization of dopamine D2 receptors which have an inhibitory action of dopamine on tumor progression. However, its clinical significance in tumor progression and prognosis remains unclear. We explored the association between STON2 and the clinicopathological characteristics of epithelial ovarian cancer (EOC). The STON2 levels in ovarian cancer and normal cell lines and tissues were detected by real-time PCR and Western blot analyses. STON2 protein expression was also detected by an immunohistochemical analysis. The clinical significance of STON2 expression in ovarian cancer was statistically analyzed. STON2 significantly increased in the ovarian cancer cell lines and tissues compared to the normal ones. In the 89 EOC samples tested, STON2 expression was significantly correlated with intraperitoneal metastasis, intestinal metastasis, intraperitoneal recurrence, ascites containing tumor cells, and CA153 level. Moreover, patients with STON2 protein overexpression were more likely to exhibit platinum resistance and to have undergone neoadjuvant chemotherapy. Patients with high STON2 protein expression had a tendency to have a shorter overall survival and a poor prognosis. A multivariate analysis showed that STON2 was an independent prognostic predictor for EOC patients. In conclusion, STON2 plays an important role in the progression and prognosis of ovarian carcinoma, especially in platinum resistance, intraperitoneal metastasis, and recurrence. STON2 can be a novel antitumor drug target and biomarker which predicts an unfavorable prognosis for EOC patients.

Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse.

Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.

Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other.

Amyloid precursor-like protein 2 (APLP2) and sortilin were reported to individually bind the proprotein convertase subtilisin/kexin type 9 (PCSK9) and regulate its activity on the low-density lipoprotein receptor (LDLR). The data presented herein demonstrate that mRNA knockdowns of APLP2, sortilin, or both in the human hepatocyte cell lines HepG2 and Huh7 do not affect the ability of extracellular PCSK9 to enhance the degradation of the LDLR. Furthermore, mice deficient in APLP2 or sortilin do not exhibit significant changes in liver LDLR or plasma total cholesterol levels. Moreover, cellular overexpression of one or both proteins does not alter PCSK9 secretion, or its activity on the LDLR. We conclude that PCSK9 enhances the degradation of the LDLR independently of either APLP2 or sortilin both ex vivo and in mice. Interestingly, when co-expressed with PCSK9, both APLP2 and sortilin were targeted for lysosomal degradation. Using chemiluminescence proximity and co-immunoprecipitation assays, as well as biosynthetic analysis, we discovered that sortilin binds and stabilizes APLP2, and hence could regulate its intracellular functions on other targets.

The possible impact of sortilin in reducing HBsAg expression in chronic hepatitis B.

Hepatitis B virus (HBV) infection is a major global health problem. Chronically infected people are at risk for progressive hepatic fibrosis and consequent cirrhosis. Hepatitis B surface antigen (HBsAg) level in serum is a complementary marker for intrahepatic HBV DNA and covalently closed circular DNA (cccDNA). Sortilin-1 (SORT1) has been reported to be involved in the post-Golgi vesicle trafficking of Apo lipoproteins degradation pathways. This study was designed to evaluate the hepatic and serum expression of HBsAg and its association with hepatic SORT1 gene expression in patients with chronic HBV. Thirty chronic hepatitis B patients with histological examination results were enrolled in this study. Liver biopsies were analyzed for hepatic HBsAg and SORT1 gene expression by immunohistochemistry and quantitative real time PCR (qRT-PCR), respectively. Twenty seven out of 30 (90%) liver biopsies had positive staining for HBsAg and showed a significant inverse association with hepatic SORT1 fold change gene expression (ß = -0.5, P = 0.042). There was significant association between HBV DNA levels and HBsAg expression in hepatocyte or serum titer of HBsAg (r = 0.39, P = 0.029; r = 0.39, P = 0.032 respectively). Serum ALT was also correlated with hepatic activity index (HAI) score (ß = 0.6, P = 0.001). Inverse association between hepatic SORT1 gene expression and hepatic HBsAg expression indicates the possible role of sortilin in HBsAg particle formation.

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus.

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet-induced obesity.

BACKGROUND & AIMS: Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS: DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS: Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS: Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Progranulin Reduced Neuronal Cell Death by Activation of Sortilin 1 Signaling Pathways After Subarachnoid Hemorrhage in Rats.

OBJECTIVES: Progranulin has been reported to have neuroprotective actions in cultured neurons. This study investigated the effect of recombinant rat progranulin on early brain injury after subarachnoid hemorrhage. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: Two hundred thirty adult male Sprague-Dawley rats weighing 280-320 g. INTERVENTIONS: Subarachnoid hemorrhage was induced in rats by endovascular perforation. Rat recombinant progranulin (1 and 3 ng) was administrated intracerebroventricularly at 1.5 hours after subarachnoid hemorrhage. Progranulin small interfering RNA was administrated by intracerebroventricularly at 1 day before subarachnoid hemorrhage induction. Subarachnoid hemorrhage grade, neurologic score, and brain water content were measured at 24 and 72 hours after subarachnoid hemorrhage. Neural apoptosis was evaluated by double immunofluorescence staining using terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick-end labeling and neuronal nuclei. For mechanistic study, the expression of progranulin, phosphorylated Akt, Akt, p-Erk, Erk, Bcl-2, and cleaved caspase-3 were analyzed by Western blot at 24 hours after subarachnoid hemorrhage. siRNA for sortilin 1 (a progranulin receptor) was used to intervene the downstream pathway. MEASUREMENTS AND MAIN RESULTS: The expression of progranulin decreased and reached the lowest point at 24 hours after subarachnoid hemorrhage. Administration of rat recombinant progranulin decreased brain water content and improved neurologic functions at both 24 and 72 hours after subarachnoid hemorrhage, while knockdown of endogenous progranulin aggravated neurologic deficits after subarachnoid hemorrhage. Rat recombinant progranulin treatment reduced neuronal apoptosis, while progranulin deficiency promoted neuronal apoptosis at 24 hours after subarachnoid hemorrhage. Rat recombinant progranulin promoted Akt activation, increased Bcl-2 level, but reduced caspase-3 level. Knockdown of progranulin binding factor sortilin 1 abolished the beneficial effects of rat recombinant progranulin at 24 hours after subarachnoid hemorrhage. CONCLUSION: Rat recombinant progranulin alleviated neuronal death via sortilin 1-mediated and Akt-related antiapoptosis pathway. Rat recombinant progranulin may have potentials to ameliorate early brain injury for subarachnoid hemorrhage patients.

Designed Modular Proteins as Scaffolds To Stabilize Fluorescent Nanoclusters.

Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.

PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress.

In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.

Dab2, megalin, cubilin and amnionless receptor complex might mediate intestinal endocytosis in the suckling rat.

We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.

Amyloid beta1₋42 (Aß42) up-regulates the expression of sortilin via the p75(NTR)/RhoA signaling pathway.

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co-receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6-month-old swedish-amyloid precursor protein/PS1dE9 transgenic mice. Aß42 enhanced the protein and mRNA expression levels of sortilin in a dose- and time-dependent manner in SH-SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75(NTR) (P75ECD-FC), or the antibody against the extracellular domain of p75(NTR), blocked the up-regulation of sortilin induced by Amyloid-ß protein (Aß), suggesting that Aß42 increased the expression level of sortilin and mRNA in SH-SY5Y via the p75(NTR) receptor. Inhibition of ROCK, but not Jun N-terminal kinase, suppressed constitutive and Aß42-induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aß42 oligomer increases sortilin gene and protein expression through p75(NTR) and RhoA signaling pathways, suggesting a potential physiological interaction of Aß42 and sortilin in Alzheimer's disease.

The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage.

BACKGROUND: Although microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively. METHODS: Subarachnoid hemorrhage was induced in TLR4-/-, TRIF-/-, MyD88-/- and wild type C57BL/6 mice by injecting 60 µl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes. RESULTS: On post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model. CONCLUSION: Our results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis.

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

miR-145 is differentially regulated by TGF-ß1 and ischaemia and targets Disabled-2 expression and wnt/ß-catenin activity.

The effect of wnt/ß-catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled-2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down-regulation of Dab2 regulates cardiac protein expression and wnt/ß-catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor-ß(1) (TGF-ß(1)). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR-145 in the 3'-UTR of Dab2. In MSC in culture, we observed that TGF-ß(1) treatment led to rapid and sustained up-regulation of pri-miR-145. Through gain and loss of function studies we demonstrate that miR-145 up-regulation was required for the down-regulation of Dab2 and increased ß-catenin activity in response to TGF-ß(1). To begin to define how Dab2 might regulate wnt/ß-catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham-operated myocardium. Following AMI, Dab2 levels were rapidly up-regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down-regulation of myocardial pri-miR-145 expression following AMI. Our data demonstrate a novel and critical role for miR-145 expression as a regulator of Dab2 expression and ß-catenin activity in response to TGF-ß(1) and hypoxia.

Immunoinhibitory adapter protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin dynamics and B cell spreading.

Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.