C1QBP Mediates Breast Cancer Cell Proliferation and Growth via Multiple Potential Signalling Pathways.

Breast carcinoma is the most prevalent cancer in women globally, with complex genetic and molecular mechanisms that underlie its development and progression. Several challenges such as metastasis and drug resistance limit the prognosis of breast cancer, and hence a constant search for better treatment regimes, including novel molecular therapeutic targets is necessary. Complement component 1, q subcomponent binding protein (C1QBP), a promising molecular target, has been implicated in breast carcinogenesis. In this study, the role of C1QBP in breast cancer progression, in particular cancer cell growth, was determined in triple negative MDA-MB-231 breast cancer cells. Depletion of C1QBP decreased cell proliferation, whereas the opposite effect was observed when C1QBP was overexpressed in MDA-MB-231 cells. Furthermore, gene expression profiling and pathway analysis in C1QBP depleted cells revealed that C1QBP regulates several signaling pathways crucial for cell growth and survival. Taken together, these findings provide a deeper comprehension of the role of C1QBP in triple negative breast cancer, and could possibly pave the way for future advancement of C1QBP-targeted breast cancer therapy.

Microglia-mediated neuroinflammation in neurodegenerative diseases.

Microglia, being the resident immune cells of the central nervous system, play an important role in maintaining tissue homeostasis and contributes towards brain development under normal conditions. However, when there is a neuronal injury or other insult, depending on the type and magnitude of stimuli, microglia will be activated to secrete either proinflammatory factors that enhance cytotoxicity or anti-inflammatory neuroprotective factors that assist in wound healing and tissue repair. Excessive microglial activation damages the surrounding healthy neural tissue, and the factors secreted by the dead or dying neurons in turn exacerbate the chronic activation of microglia, causing progressive loss of neurons. It is the case observed in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. This review gives a detailed account of the microglia-mediated neuroinflammation in various neurodegenerative diseases. Hence, resolving chronic inflammation mediated by microglia bears great promise as a novel treatment strategy to reduce neuronal damage and to foster a permissive environment for further regeneration effort.

Epigenetic regulation of microglial phosphatidylinositol 3-kinase pathway involved in long-term potentiation and synaptic plasticity in rats.

Microglia are the main form of immune defense in the central nervous system. Microglia express phosphatidylinositol 3-kinase (PI3K), which has been shown to play a significant role in synaptic plasticity in neurons and inflammation via microglia. This study shows that microglial PI3K is regulated epigenetically through histone modifications and posttranslationally through sumoylation and is involved in long-term potentiation (LTP) by modulating the expression of brain-derived neurotrophic factor (BDNF), which has been shown to be involved in neuronal synaptic plasticity. Sodium butyrate, a histone deacetylase inhibitor, upregulates PI3K expression, the phosphorylation of its downstream effectors, AKT and cAMP response element-binding protein (CREB), and the expression of BDNF in microglia, suggesting that BDNF secretion is regulated in microglia via epigenetic regulation of PI3K. Further, knockdown of SUMO1 in BV2 microglia results in a decrease in the expression of PI3K, the phosphorylation of AKT and CREB, as well as the expression of BDNF. These results suggest that microglial PI3K is epigenetically regulated by histone modifications and posttranslationally modified by sumoylation, leading to altered expression of BDNF. Whole-cell voltage-clamp showed the involvement of microglia in neuronal LTP, as selective ablation or disruption of microglia with clodronate in rat hippocampal slices abolished LTP. However, LTP was rescued when the same hippocampal slices were treated with active PI3K or BDNF, indicating that microglial PI3K/AKT signaling contributes to LTP and synaptic plasticity. Understanding the mechanisms by which microglial PI3K influences synapses provides insights into the ways it can modulate synaptic transmission and plasticity in learning and memory.

Transcriptome analysis reveals intermittent fasting-induced genetic changes in ischemic stroke.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

Nanoparticle-Based Technology Approaches to the Management of Neurological Disorders.

Neurological disorders are the most devastating and challenging diseases associated with the central nervous system (CNS). The blood-brain barrier (BBB) maintains homeostasis of the brain and contributes towards the maintenance of a very delicate microenvironment, impairing the transport of many therapeutics into the CNS and making the management of common neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebrovascular diseases (CVDs) and traumatic brain injury (TBI), exceptionally complicated. Nanoparticle (NP) technology offers a platform for the design of tissue-specific drug carrying systems owing to its versatile and modifiable nature. The prospect of being able to design NPs capable of successfully crossing the BBB, and maintaining a high drug bioavailability in neural parenchyma, has spurred much interest in the field of nanomedicine. NPs, which also come in an array of forms including polymeric NPs, solid lipid nanoparticles (SLNs), quantum dots and liposomes, have the flexibility of being conjugated with various macromolecules, such as surfactants to confer the physical or chemical property desired. These nanodelivery strategies represent potential novel and minimally invasive approaches to the treatment and diagnosis of these neurological disorders. Most of the strategies revolve around the ability of the NPs to cross the BBB via various influx mechanisms, such as adsorptive-mediated transcytosis (AMT) and receptor-mediated transcytosis (RMT), targeting specific biomarkers or lesions unique to that pathological condition, thereby ensuring high tissue-specific targeting and minimizing off-target side effects. In this article, insights into common neurological disorders and challenges of delivering CNS drugs due to the presence of BBB is provided, before an in-depth review of nanoparticle-based theranostic strategies.

Activation of microglia in acute hippocampal slices affects activity-dependent long-term potentiation and synaptic tagging and capture in area CA1.

Activity dependent setting of synaptic tags is critical for the establishment and maintenance of long-term plasticity and its associative properties such as synaptic tagging and capture (STC), a widely studied cellular model of associative memory. Although the known mechanisms of STC such as setting of synaptic tags or distribution of plasticity related proteins (PRPs) are the processes mainly happening within the neuronal compartments, the role of non-neuronal components is still elusive. Here, we report that microglia has a specific role in setting the synaptic tags and thus promotes long-term plasticity and STC. Treatment of hippocampal slices with clodronate, a specific inhibitor of microglia, resulted in an activated morphology of microglia but not of the hippocampal pyramidal neurons, oligodendrocytes or astrocytes. Activation of microglia before or 60 min after the induction of long-term plasticity prevented its maintenance and thus the expression of STC. Interestingly, activation of microglia 2 h after the induction of long-term plasticity neither prevented its maintenance nor its associative interaction with activated nearby synaptic populations. Given the half-life of synaptic tags is until about 60-90 min, activation of microglia beyond this time point while the maintenance phase is still unperturbed, suggests a lack of microglial interference in the synthesis or trigger of plasticity related products. Thus, our study provides the first evidence that microglia play a critical role in the setting of synaptic tags during the early phase of activity dependent plasticity.

Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke.

Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke - a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions.

Paracrine effects of mesenchymal stem cells-conditioned medium on microglial cytokines expression and nitric oxide production.

BACKGROUND/AIM: Microglia, the resident macrophages in the central nervous system, secrete various proinflammatory cytokines and undergo proliferation upon activation in various neurodegenerative diseases. Activation of microglia has been implicated in exacerbation of various neurodegenerative diseases. Recently, it has been proposed that mesenchymal stem cells (MSC) have immunosuppressive properties and the potential to moderate inflammation. This study aimed to elucidate the effects of MSC-conditioned medium (MSC-CM) in modulating microglial activation by analyzing microglial proinflammatory and anti-inflammatory factors [interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and IL-10], signaling pathway molecules [NFκB, c-Jun N-terminal kinase (JNK) and MKP-1) and NO production. METHODS: Immortalized murine microglia cell line, BV2 microglia and primary microglia isolated from C57BL/6 mouse pup brains were used in this study. Mouse MSC were isolated from the male C57BL/6 mouse tibia and fibula. The effects of MSC-CM on the expression of inflammatory cytokines and signaling molecules in microglia were elucidated using RT-PCR, immunofluorescence analysis and Western blot analysis. NO production in microglia was assessed using a Griess kit. RESULTS: MSC-CM significantly reduced the mRNA and protein expression levels of proinflammatory cytokines (IL-6 and TNF-α) in microglia activated by lipopolysaccharide (LPS). In addition, MSC-CM significantly reduced the protein expression of NFκB, JNK and c-Jun, but increased the expression levels of IL-10 and MKP-1 in activated BV2 microglia. NO production and iNOS expression by BV2 microglia in MSC-CM were increased. CONCLUSIONS: Overall, our findings suggest that MSC immunomodulate microglial activities through paracrine effects.

microRNA-200b modulates microglia-mediated neuroinflammation via the cJun/MAPK pathway.

Chronic activation of microglia, the macrophages of the CNS, has been shown to enhance neuronal damage because of excessive release of proinflammatory cytokines and neurotoxic molecules in a number of neurodegenerative diseases. Recent reports showed altered microRNA (miRNA) expression in immune-mediated pathologies, thus suggesting that miRNAs modulate expression of genes involving immune responses. This study demonstrates that miRNA-200b is expressed in microglia and modulates inflammatory response of microglia by regulating mitogen-activated protein kinase pathway. miRNA-200b expression was found to be down-regulated in activated microglia in vivo (traumatic brain injury rat model) and in vitro. A luciferase assay and loss- and gain-of-function studies revealed c-Jun, the transcription factor of cJun-N terminal kinase (JNK) mitogen-activated protein kinase pathway to be the target of miR-200b. Knockdown of miR-200b in microglia increased JNK activity along with an increase in pro-inflammatory cytokines, inducible nitric oxide synthase expression and nitric oxide (NO) production. Conversely, over-expression of miRNA-200b in microglia resulted in a decrease in JNK activity, inducible nitric oxide synthase expression, NO production and migratory potential of activated microglia. Furthermore, miR-200b inhibition resulted in increased neuronal apoptosis after treatment of neuronal cells with conditioned medium obtained from microglial culture. Taken together, these results indicate that miRNA-200b modulates microglial inflammatory process including cytokine secretion, NO production, migration and neuronal survival.

Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer.

Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia.

BACKGROUND: Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury. METHODS: One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining. RESULTS: TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1ß and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1ß, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1ß and iNOS on microglia post-hypoxia. CONCLUSION: Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.

Maternal Diabetes Deregulates the Expression of Mecp2 via miR-26b-5p in Mouse Embryonic Neural Stem Cells.

Maternal diabetes has been associated with a greater risk of neurodevelopmental disorders in offspring. It has been established that hyperglycemia alters the expression of genes and microRNAs (miRNAs) regulating the fate of neural stem cells (NSCs) during brain development. In this study, the expression of methyl-CpG-binding protein-2 (Mecp2), a global chromatin organizer and a crucial regulator of synaptic proteins, was analyzed in NSCs obtained from the forebrain of embryos of diabetic mice. Mecp2 was significantly downregulated in NSCs derived from embryos of diabetic mice when compared to controls. miRNA target prediction revealed that the miR-26 family could regulate the expression of Mecp2, and further validation confirmed that Mecp2 is a target of miR-26b-5p. Knockdown of Mecp2 or overexpression of miR-26b-5p altered the expression of tau protein and other synaptic proteins, suggesting that miR-26b-5p alters neurite outgrowth and synaptogenesis via Mecp2. This study revealed that maternal diabetes upregulates the expression of miR-26b-5p in NSCs, resulting in downregulation of its target, Mecp2, which in turn perturbs neurite outgrowth and expression of synaptic proteins. Overall, hyperglycemia dysregulates synaptogenesis that may manifest as neurodevelopmental disorders in offspring from diabetic pregnancy.

Transcriptome analysis of amoeboid and ramified microglia isolated from the corpus callosum of rat brain.

BACKGROUND: Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. RESULTS: To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC. CONCLUSIONS: The novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis.

Protein O-GlcNAcylation is a dynamic, nutrient-sensitive mono-glycosylation deposited on numerous nucleo-cytoplasmic and mitochondrial proteins, including transcription factors, epigenetic regulators, and histones. However, the role of protein O-GlcNAcylation on epigenome regulation in response to nutrient perturbations during development is not well understood. Herein we recapitulated early human embryonic neurogenesis in cell culture and found that pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells' neuronal differentiation leads to up-regulation of key neurogenic transcription factor genes. This transcriptional de-repression is associated with reduced H3K27me3 and increased H3K4me3 levels on the promoters of these genes, perturbing promoter bivalency possibly through increased EZH2-Thr311 phosphorylation. Elevated O-GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112O-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated O-GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of O-GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate O-GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

A comparative investigation on the mechanical properties and cytotoxicity of Cubic, Octet, and TPMS gyroid structures fabricated by selective laser melting of stainless steel 316L.

Metallic lattice structures can be fabricated by selective laser melting (SLM) with purposefully designed pores and controlled pore sizes that can bio mimic the natural bone, providing adequate mechanical and biological support for the patients. Strut-based structures, like Cubic, Octet; and sheet-based structures, like triply periodic minimal surface (TPMS) gyroid, have been studied extensively in the past. However, it lacks enough comparative study on the mechanical properties and cytotoxicity among these structures. Therefore, Cubic, Octet, and TPMS gyroid of Stainless steel 316 L (SS316L) are designed, manufactured, and characterized at 40/50/60% relative densities in this study. Moreover, the flowability, density characteristics, and cytotoxicity of SS316L powder are validated to ascertain its suitability for 3D printing and implant application. Based on refining the Gibson-Ashby model, it is possible to predict or design the mechanical properties via adjusting the relative densities. The results indicate these structures demonstrated appropriate Young's modulus and outstanding biocompatibility.

A biomechanical evaluation on Cubic, Octet, and TPMS gyroid Ti6Al4V lattice structures fabricated by selective laser melting and the effects of their debris on human osteoblast-like cells.

Lattice structures are widely used in orthopedic implants due to their unique features, such as high strength-to-weight ratios and adjustable biomechanical properties. Based on the type of unit cell geometry, lattice structures may be classified into two types: strut-based structures and sheet-based structures. In this study, strut-based structures (Cubic & Octet) and sheet-based structure (triply periodic minimal surface (TPMS) gyroid) were investigated. The biomechanical properties of the three different Ti6Al4V lattice structures fabricated by selective laser melting (SLM) were investigated using room temperature compression testing. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were used to check the 3D printing quality with regards to defects and quantitative compositional information of 3D printed parts. Experimental results indicated that TPMS gyroid has superior biomechanical properties when compared to Cubic and Octet. Also, TPMS gyroid was found to be less affected by the variations in relative density. The biocompatibility of Ti6Al4V lattice structures was validated through the cytotoxicity test with human osteoblast-like SAOS2 cells. The debris generated during the degradation process in the form of particles and ions is among the primary causes of implant failure over time. In this study, Ti6Al4V particles with spherical and irregular shapes having average particle sizes of 36.5 µm and 28.8 µm, respectively, were used to mimic the actual Ti6Al4V particles to understand their harmful effects better. Also, the effects and amount of Ti6Al4V ions released after immersion within the cell culture media were investigated using the indirect cytotoxicity test and ion release test.

Stage- and Subfield-Associated Hippocampal miRNA Expression Patterns after Pilocarpine-Induced Status Epilepticus.

OBJECTIVE: To investigate microRNA (miRNA) expression profiles before and after pilocarpine-induced status epilepticus (SE) in the cornu ammonis (CA) and dentated gyrus (DG) areas of the mouse hippocampus, and to predict the downstream proteins and related pathways based on bioinformatic analysis. METHODS: An epileptic mouse model was established using a pilocarpine injection. Brain tissues from the CA and DG were collected separately for miRNA analysis. The miRNAs were extracted using a kit, and the expression profiles were generated using the SurePrint G3 Mouse miRNA microarray and validated. The intersecting genes of TargetScan and miRanda were selected to predict the target genes of each miRNA. For gene ontology (GO) studies, the parent-child-intersection (pci) method was used for enrichment analysis, and Benjamini-Hochberg was used for multiple test correction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to detect disease-related pathways among the large list of miRNA-targeted genes. All analyses mentioned above were performed at the time points of control, days 3, 14, and 60 post-SE. RESULTS: Control versus days 3, 14, and 60 post-SE: in the CA area, a total of 131 miRNAs were differentially expressed; 53, 49, and 26 miRNAs were upregulated and 54, 10, and 22 were downregulated, respectively. In the DG area, a total of 171 miRNAs were differentially expressed; furthermore, 36, 32, and 28 miRNAs were upregulated and 78, 58, and 44 were downregulated, respectively. Of these, 92 changed in both the CA and DG, 39 only in the CA, and 79 only in the DG area. The differentially expressed miRNAs target 11-1630 genes. Most of these proteins have multiple functions in epileptogenesis. There were 15 common pathways related to altered miRNAs: nine different pathways in the CA and seven in the DG area. CONCLUSIONS: Stage- and subfield-associated hippocampal miRNA expression patterns are closely related to epileptogenesis, although the detailed mechanisms need to be explored in the future.

Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats.

Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

Dexamethasone inhibits the Nox-dependent ROS production via suppression of MKP-1-dependent MAPK pathways in activated microglia.

BACKGROUND: Nox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear. RESULTS: The present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release. CONCLUSION: In conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.