Hypoxia Regulated Gene Network in Glioblastoma Has Special Algebraic Topology Structures and Revealed Communications Involving Warburg Effect and Immune Regulation Abstract Hypoxia regulated genes (HRGs) formed a complex molecular interaction network (MINW), contributing to many aspects of glioblastoma (GBM) tumor biology. However, little is known about the intrinsic structures of the HRGs–MINW, mainly due to a lack of analysis tools to decipher MINWs. By introducing general hyper-geometric distribution, we obtained a statistically reliable gene set of HRGs (SR-HRGs) from several datasets. Next, MINWs were reconstructed from several independent GBM expression datasets. Algebraic topological analysis was performed to quantitatively analyze the amount of equivalence classes of cycles in various dimensions by calculating the Betti numbers. Persistent homology analysis of a filtration of growing networks was further performed to examine robust topological structures in the network by investigating the Betti curves, life length of the cycles. Random networks with the same number of node and edge and degree distribution were produced as controls. As a result, GBM–HRGs–MINWs reconstructed from different datasets exhibited great consistent Betti curves to each other, which were significantly different from that of random networks. Furthermore, HRGs–MINWs reconstructed from normal brain expression datasets exhibited topological structures significantly different from that of GBM–HRGs–MINWs. Analysis of cycles in GBM–HRGs–MINWs revealed genes that had clinical implications, and key parts of the cycles were also identified in reconstructed protein–protein interaction networks. In addition, the cycles are composed by genes involved in the Warburg effect, immune regulation, and angiogenesis. In summary, GBM–HRGs–MINWs contained abundant molecular interacting cycles in different dimensions, which are composed by genes involved in multiple programs essential for the tumorigenesis of GBM, revealing novel interaction diagrams in GBM and providing novel potential therapeutic targets.

Neuroprotective Effect of Natural Alkaloid Fangchinoline Against Oxidative Glutamate Toxicity: Involvement of Keap1-Nrf2 Axis Regulation Abstract Oxidative glutamate toxicity plays a vital role in the neurodegeneration diseases, including Alzheimer’s diseases (AD). This study set out with the aim to investigate the beneficial effects of fangchinoline (FAN), a natural alkaloid, against glutamate-induced oxidative damage, and to clarify the underlying cellular and biochemical mechanisms. FAN prevented HT22 cells death from oxidative glutamate cytotoxicity in a dose-dependent manner, and significantly attenuated the overproduction of intracellular reactive oxygen species (ROS) and reversed the reduction of superoxide dismutase (SOD) activity induced by glutamate. Further investigations on the underlying mechanisms demonstrated that FAN potently up-regulated the protein level of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase (HO-1), in glutamate-exposed HT22 cells. The protective effects of FAN were almost completely antagonized by inhibitor of Nrf2. Subsequent studies revealed that FAN could down-regulate Kelch-like ECH-associated protein 1 (Keap1) in both mRNA level and protein level. To sum up, our result demonstrated the protective effects of FAN against glutamate-induced oxidative neuronal damage, and for the first time clarified the anti-oxidative mechanisms of FAN involve activating endogenous antioxidant defense system including enhancing SOD activity and regulating Keap1/Nrf-2 antioxidation signaling through modulation of Keap1 expression. Above results shed more light on the molecular mechanisms of FAN’s neuroprotective effects, and may provide important clues for the drug development in preventing oxidative stress-associated neurodegenerative diseases.

CeO 2 @PAA-LXW7 Attenuates LPS-Induced Inflammation in BV2 Microglia Abstract Microglia are the inherent immune effector cells in the central nervous system (CNS), are activated rapidly when the CNS is stimulated by ischaemia, infection, injury, etc. and participate in and aggravate the development of inflammatory reactions in the CNS. During the process of microglial activation, inflammatory factors such as TNF-α and IL-1β and an abundance of reactive oxygen species (ROS)/reactive nitrogen species (RNS), are released by damaged nerve cells. LXW7 is a small molecule peptide and specifically binds with integrin αvβ3. Cerium oxide nanoparticles (nanoceria) are strong free radical scavengers and are widely used in many studies. In this research, a model of inflammation was established using lipopolysaccharide (LPS) to induce BV2 microglia activation, and the effects of CeO2@PAA (synthetic nanoscale cerium oxide particles), LXW7 and CeO2@PAA-LXW7 were evaluated. We detected the expression level of inflammatory factors, the release of NO in BV2 cells and the generation of intracellular ROS. The expression levels of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) and their phosphorylated proteins were detected in BV2 microglia. We found that CeO2@PAA, LXW7 and CeO2@PAA-LXW7 all effectively inhibited the activation of BV2 microglia, reduced the production of cytokines and the release of NO and reduced the production of intracellular ROS. The three treatments all inhibited the phosphorylation of FAK and STAT3 in BV2 microglia. Regarding these effects, CeO2@PAA-LXW7 was more effective than the other two monotherapies. Our data indicate that CeO2@PAA, LXW7 and CeO2@PAA-LXW7 can exert a neuroprotective function by inhibiting the inflammatory response of LPS-induced BV2 microglia. LXW7 may inhibit the activation of FAK and STAT3 signals in combination with integrin αvβ3 to restrain neuroinflammation and the antioxidative stress effect of cerium oxide; hence, CeO2@PAA-LXW7 can exert a more robust anti-inflammatory and neuroprotective effect via synergistically suppressing the ability of LXW7 to influence the integrin pathway and the free radical-scavenging ability of CeO2@PAA.

Prognostic Value of Plasma Trimethylamine N-Oxide Levels in Patients with Acute Ischemic Stroke Abstract Trimethylamine N-oxide (TMAO) has emerged as a newly identified gut microbiota-dependent metabolite contributing to a variety of diseases, such as diabetes, atherosclerosis, and cardiovascular diseases. The aim of our study was to determine whether a relatively high TMAO level is associated with an increased risk of poor outcome in ischemic stroke patients. From June 2018 to December 2018, we prospectively recruited acute ischemic stroke patients diagnosed within 24 h of symptom onset. The plasma TMAO level was measured at admission for all patients. Functional outcome was evaluated at 3 months after the stroke using the modified Rankin Scale (mRS) and then dichotomized as favorable (mRS 0–2) or unfavorable (mRS 3–6). A multivariate logistic regression analysis was conducted to evaluate the association between TMAO concentration and poor functional outcome and mortality at 3 months. Of the 225 acute ischemic stroke patients included in the analysis, the median TMAO concentration was 3.8 µM (interquartile range, 1.9–4.8 µM). At 3 months after admission, poor functional outcome was observed in 116 patients (51.6%), and 51 patients had died (22.7%). After adjusting for potential confounders, patients with TMAO levels in the highest quartile were more likely to have higher risks of poor functional outcome [compared with the lowest quartile, odds ratio (OR) 3.63; 95% confidence interval (CI) 1.34–9.82; P = 0.011] and mortality (OR 4.27; 95% CI 1.07–17.07; P = 0.040). Our data suggest that a high plasma TMAO level upon admission may predict unfavorable clinical outcomes in acute ischemic stroke patients.

Ca 2+ Signaling in Oligodendrocyte Development Abstract Calcium signaling has essential roles in the development of the nervous system, from neural induction to the proliferation, migration, and differentiation of both neuronal and glia cells. The temporal and spatial dynamics of Ca2+ signals control the highly diverse yet specific transcriptional programs that establish the complex structures of the nervous system. Ca2+-signaling pathways are shaped by interactions among metabotropic signaling cascades, ion channels, intracellular Ca2+ stores, and a multitude of downstream effector proteins that activate specific genetic programs. Progress in the last decade has led to significant advances in our understanding of the functional architecture of Ca2+ signaling networks involved in oligodendrocyte development. In this review, we summarize the molecular and functional organizations of Ca2+-signaling networks during the differentiation of oligodendrocyte, especially its impact on myelin gene expression, proliferation, migration, and myelination. Importantly, the existence of multiple routes of Ca2+ influx opens the possibility that the activity of calcium channels can be manipulated pharmacologically to encourage oligodendrocyte maturation and remyelination after demyelinating episodes in the brain.

The PILRA G78R Variant Correlates with Higher HSV-1-Specific IgG Titers in Alzheimer’s Disease Abstract Alzheimer’s disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognitive performance; Mild Cognitive Impairment (MCI) is instead an objective decline in cognitive performance that does not reach pathology. Paired immunoglobulin-like type 2 receptor alpha (PILRA) is a cell surface inhibitory receptor that was recently suggested to be involved in AD pathogenesis. In particular, the arginine-to-glycine substitution in position 78 (R78, rs1859788) was shown to be protective against AD. Herpes simplex virus type 1 (HSV-1) infection is suspected as well to be involved in AD. Interestingly, HSV-1 uses PILRA to infect cells, and HSV-1 infects more efficiently PIRLA G78 compared to R78 macrophages. We analyzed PILRA rs1859788 polymorphism and HSV-1 humoral immune responses in AD (n = 61) and MCI patients (n = 48), and in sex and age matched healthy controls (HC; n = 57). The rs1859788 PILRA genotype distribution was similar among AD, MCI and HC; HSV-1 antibody (Ab) titers were increased in AD and MCI compared to HC (p < 0.05 for both comparisons). Notably, HSV-1-specific IgG1 were significantly increased in AD patients carrying PILRA R78 rs1859788 AA than in those carrying G78 AG or GG (p = 0.01 for both comparisons), and the lowest titers of HSV-1-specific IgG1 were observed in rs1859788 GG AD. HSV-1 IgG are increased in AD patients with the protective R78 PILRA genotype. Because in AD patients brain atrophy is inversely correlated with HSV-1-specific IgG titers, results herein suggest a possible link between two important genetic and infective factors suspected to be involved in AD pathogenesis.

Estrogen Receptors Alpha and Beta in POA-AHA Region Regulate Asymmetrically Ovulation Abstract We examined the role of the estrogen receptors alpha (ERα) and beta (ERβ) in of the preoptic-anterior hypothalamic area (POA-AHA) in the regulation of ovulation in rats. The number of ERα- and ERβ-immunoreactive (-ir) cells was determined at 09:00, 13:00, and 17:00 h of each stage of the estrous cycle in intact rats. Additionally, the effects of blocking ERα and ERβ on ovulation rate at 09:00 h on diestrus-2 or proestrus day through the microinjection of methyl-piperidino-pyrazole (MPP) or cyclofenil in either side of POA-AHA were evaluated. The number of ERα-ir and ERβ-ir cells in POA-AHA varied in each phase of estrous cycle. Either MPP or cyclofenil in the right side of POA-AHA on diestrus-2 day reduced the ovulation rate, while at proestrus day it was decreased in rats treated in either side with MPP, and in those treated with cyclofenil in the left side. MPP or cyclofenil produced a decrease in the surge of luteinizing hormone levels (LH) and an increase in progesterone and follicle stimulating hormone (FSH). Replacement with synthetic luteinizing hormone-releasing hormone in non-ovulating rats treated with MPP or cyclofenil restored ovulation. These results suggest that activation of estrogen receptors on the morning of diestrus-2 and proestrus day asymmetrically regulates ovulation and appropriately regulates the secretion of FSH and progesterone in the morning and afternoon of proestrus day. This ensures that both, the preovulatory secretion of LH and ovulation, occur at the right time.

KDELR2 Promotes Glioblastoma Tumorigenesis Targeted by HIF1a via mTOR Signaling Pathway Abstract The KDEL (Lys-Asp-Glu-Leu) receptors (KDELRs), proteins with seven transmembrane domains, are primarily responsible for endoplasmic reticulum (ER) homeostasis. Recent studies have found additional function of KDELRs in growth, cellular secretory traffic, immune response, and autophagy; however, its role in tumorigenesis is still poorly understood. Here, we showed that KDELR2 is highly expressed in glioblastoma (GBM) tissues. Reviewing the expression of KDELR2 in TCGA and REMBRANDT database, we found that higher expression of KDELR2 is associated with shorter survival of GBM patients. We explored the effect of KDELR2 on tumorigenesis in GBM cells and animal model (nude mice), and identified KDELR2 as oncogene promoting cell proliferation. Additionally, KDELR2 expression in GBM cells correlated positively with HIF1alpha (HIF1α) expression, and we demonstrated by ChIP-qPCR and luciferase reporter assay that the upstream region of the KDELR2 gene is directly targeted by HIF1alpha. Taken together, our data suggest that KDELR2 is a target gene downstream of HIF1-alpha driving the malignancy of GBM and could eventually serve as a therapeutic target for the treatment of GBM patients.

Lidocaine Potentiates SOCS3 to Attenuate Inflammation in Microglia and Suppress Neuropathic Pain Abstract Lidocaine is one of the typical local anesthetics that are frequently used in the peripheral nerve blocks and pain management. Emerging evidence have shown that lidocaine may exert anti-inflammatory effect involving neuropathic pain. However, the effect and underlying mechanism of lidocaine in suppressing neuroinflammation in neuropathic pain are incompletely revealed. In this study, effects of lidocaine on the suppressors of cytokine-signaling protein 3 (SOCS3) in microglia are investigated in chronic constriction injury (CCI) rat model and lipopolysaccharide (LPS)-stimulated BV-2 cells. It was shown that intrathecal injection of lidocaine substantially alleviated CCI-induced neuropathic pain, as reflected by the decreased thermal latency and mechanical threshold. Lidocaine reduced the CCI-evoked spinal injury and cell apoptosis. CCI induced an significant increase of IBA1+ microglia accompanied by the increase of inflammatory cytokines IL-6 and IL-1β, which were suppressed after lidocaine administration. SOCS3 expression in IBA1+ microglia was notably upregulated in response to lidocaine injection, which presented in a similar pattern in LPS-activated BV-2 cells. Furthermore, lidocaine upregulated SOCS3 expression dependent of pCREB, and CREB silencing greatly discounted this effect. The intrathecal injection of lentiviral vectors LV-SOCS3 efficiently alleviated CCI-evoked neuropathic pain and reduced spinal IBA1+ microglia. SOCS3 overexpression contributed to the inhibition of neuroinflammation by decreasing the expression and activation of p38 MAPK and NF-κB stimulated by LPS. Collectively, lidocaine promoted the SOCS3 expression in microglia, in turn leading to suppression of IBA1+ microglia accumulation and p38 MAPK and NF-κB, which may expand our understanding on lidocaine in suppressing neuroinflammation and neuropathic pain.

SIRT1 Activation Attenuates Bone Cancer Pain by Inhibiting mGluR1/5 Abstract Bone cancer pain (BCP), which is induced by primary or metastatic bone cancer, remains a clinically challenging problem due to the poor understanding of its mechanisms. Sirtuin 1 (SIRT1) plays an important role in various pain models. Intrathecal administration of SRT1720, a SIRT1 activator, attenuates BCP in a rat model. However, the expression and activity of SIRT1 during the development and maintenance of BCP remain unknown. Furthermore, the underlying mechanism of SIRT1 in BCP remains ambiguous. In this study, we detected the time course of SIRT1 expression and activity in the spinal cord of mice with BCP and examined whether SRT1720 alleviated BCP by inhibiting metabotropic glutamatergic receptor (mGluR) 1/5 expression. In addition, we downregulated spinal SIRT1 expression in normal mice through an intrathecal injection of AAV-SIRT1-shRNA and then assessed pain behavior and mGluR1/5 expression. Mice with BCP developed significant mechanical allodynia and spontaneous flinching, accompanied by decreased levels of the SIRT1 protein, mRNA, and activity in the spinal cord. The SRT1720 treatment produced an analgesic effect on tumor-bearing mice and decreased the spinal levels of the mGluR1/5 protein and mRNA. In contrast, the AAV-SIRT1-shRNA treatment induced pain behavior in normal mice and increased the spinal levels of the mGluR1/5 protein and mRNA. The results suggested a critical role for SIRT1 in the development and maintenance of BCP and further indicated that activation of SIRT1 in the spinal cord by SRT1720 functionally reverses BCP in mice by inhibiting mGluR1/5.