Xiaolu Deng

Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier

PURPOSE The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of human intestinal epithelial cell induced by tumor necrosis factor-alpha (TNF-α). METHODS To confirm that TNF-α induces epithelial barrier hyperpermeability by disrupting tight junction, Caco-2 cells were exposed to TNF-α, and changes in epithelial permeability (via TER assay), F-actin dynamics (via Rhodamine-phalloidin staining) and tight junction protein expression (via western blot) were monitored. Moreover, to ensure that NF-κB participated in the regulatory mechanisms, Caco-2 cells were transfected with DNMu-IκBα or control plasmids, the above experiments were repeated and the activation effect of TNF-α on NF-κB was detected by luciferase reporter assays. Lastly, we took dominant negative plasmid and knockdown approaches to investigate the potential importance of the NF-κB/myosin light chain kinase (MLCK)/myosin light chain phosphorylation (pMLC) pathways in TNF-a-mediated damage. RESULT TNF-α could cause NF-κB activation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were alleviated by inhibiting NF-κB. TNF-α induced increase of MLCK transcription and MLC phosphorylation act later than NF-κB activation, which could be suppressed both by inactivating and deleting NF-κB. CONCLUSIONS TNF-α induces intestinal epithelial cell hyperpermeability by disrupting TJs, in part through MLCK upregulation, in which NF-κB is the positive upstream regulator for MLCK.

Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

BackgroundTumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC) isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC) barrier dysfunction.MethodsBend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL). RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER). p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability.ResultsWe observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA) transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER.ConclusionsTaken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF-α signaling to RhoA, and that this plays a critical role in signaling F-actin rearrangement and barrier dysfunction in BMECs.

RhoA and NF-κB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability.

PURPOSE The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of brain microvascular cell lines induced by lipopolysaccharide (LPS). METHODS To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3 cells were exposed to LPS, and changes in endothelial permeability (transendothelial electrical resistance (TEER) assay), F-actin dynamics (Rhodamine-Phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. Moreover, to ensure that both RhoA and NF-κB participated in the regulatory mechanisms, Bend.3 cells were transfected with n19RhoA and DNMu-IκBα plasmids, and the above experiments were repeated. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in transfected and untreated Bend.3 cells. Lastly, to investigate whether RhoA and NF-κB regulate MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation in untreated and transfected Bend.3 cells by western blot. RESULT LPS caused RhoA and NF-κB activation, MLC phosphorylation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were suppressed by inhibitors of RhoA or NF-κB; inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-κB activation, but the inverse was not true. CONCLUSIONS LPS induces brain microvascular endothelial barrier hyperpermeability by disrupting TJs, in part through RhoA and NF-κB activation, in which RhoA is the positive upstream regulator for NF-κB.

Myoloid-related protein 8, an endogenous ligand of Toll-like receptor 4, is involved in epileptogenesis of mesial temporal lobe epilepsy via activation of the nuclear factor-κB pathway in astrocytes.

The role of Toll-like receptor 4 (TLR4) in the activation of innate immunity has been extensively studied in the past several years. Here, we are the first to report that myeloid-related protein 8 (MRP8), an endogenous TLR4 ligand, is involved in the epileptogenesis of mesial temporal lobe epilepsy (MTLE). We find that the expression of MRP8, TLR4, and interleukin 1-β (IL-1β) was upregulated in a MTLE model during both acute and chronic disease stages. We next investigated the possible roles played by astrocytes, which have been shown to be the major source of IL-1β during epilepsy. Stimulation via MRP8 led to the induction of IL-1β in astrocytes in vitro, accompanied by the activation of Nuclear Factor-κB, while knockdown of TLR4 or inhibition of NF-κB in astrocytes prevented this IL-1β induction. Thus, MRP8 may potentiate the perpetuation of MTLE by activating the NF-κB pathway in astrocytes, and could be a new target for anticonvulsant therapies.

Leukodystrophy associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFAF1 gene

Abstract Mitochondrial energy metabolism disorder is one of the important reasons of leukodystrophy. Mutations of mitochondrial complex I genes have been implicated in more common neurological disorders such as Leigh syndrome. We describe a case of a child manifested as regression of mental and motor development, aggravated obviously after suffering infection. Physical and auxiliary examinations demonstrated that a series of changes including white matter lesions of magnetic resonance imaging, peripheral neuropathy with high muscle tension and hyperreflexia of limbs pointed to the diagnosis of leukodystrophy, with what can’t explain the high levels of lactate and creatine kinase. Spontaneously, genetic analysis covered known leukodystrophy and mitochondrial genes were adapted for this child and his parents. Results showed the child was compound heterozygous mutation (c.278A > G; c.247G > A) within exon 2 in the NDUFAF1 gene, his parents carried a heterozygous mutation each. The authors report a case of leukodystrophy associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFAF1 gene. This is the first report that NDUFAF1 mutations cause leukodystrophy.

The role of ubiquitin/Nedd4-2 in the pathogenesis of mesial temporal lobe epilepsy

Although the pathogenesis and epileptogenesis of mesial temporal lobe epilepsy (MTLE) have been studied for years, many questions remain. The ubiquitin-proteasome system (UPS) is one factor that might regulate ion channels, inflammation and neuron excitability. Nedd4-2 is an E3 ubiquitin ligase linked with ion channels and synaptic vesicle recycling. Here, we explore the role of the UPS and its E3 ligase Nedd4-2 in the pathogenesis of MTLE. Our western blot results revealed that ubiquitin and Nedd4-2 were expressed differentially in different stages of MTLE. Co-immunoprecipitation and double immunostaining results indicated that Nedd4-2 was the substrate protein of ubiquitin both in vivo and in vitro. Inhibition of the UPS aggravated the epileptogenesis of MTLE, causing early and frequent spontaneous seizures, more obvious neuron loss and aberrant mossy fiber sprouting. Inhibition of ubiquitin also enhanced the activation of Nedd4-2, and switched ion channel α-ENaC downstream. Our study is the first to report that the UPS participates in the pathogenesis of MTLE, inhibition of UPS could aggravate the epileptogenesis, and that Nedd4-2 is a critical E3 ligase involved in this process.

Molecular mechanism for change in permeability in brain microvascular endothelial cells induced by LPS.

OBJECTIVE To investigate the molecular mechanism for change in permeability in brain microvascular endothelial cells (bEnd.3) induced by lipopolysaccharide (LPS). METHODS Monolayers of bEnd.3 were exposed to LPS, in the presence or absence of exoenzyme C3 transferase. We monitored the monolayer barrier integrity by transendothelial electrical resistance assay (TEER), activity of RhoA by pull down assay, NF-κB by luciferase reporter assay, and F-actin dynamic structure by Rhodamine-phalloidin staining. RESULTS Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the normal condition, the average TEER of bEnd.3 was (82.33±3.11) ω.cm², while it decreased apparently to (53.67±2.01) ω.cm² and (37.67±3.05) ω.cm² when the cells had been treated for 3 and 12 h with LPS (P<0.05). Such effects could be inhibited partly by pretreatment of RhoA inhibitor exoenzyme C3 transferase. LPS activated RhoA and NF-κB at 0.5 h. The C3 transferase could significantly reverse the NF-κB activation (P<0.05). The F-actin rearrangements displayed in a time-dependent manner and occurred originally after the stimulation of LPS for 3 h, which could be diluted by the pretreatment of C3 transferase as well. CONCLUSION LPS induces the disruption of F-actin cytoskeleton and brain microvascular endothelial barrier integrity, in part, through RhoA and NF-κB activation. The mechanism underlying this pathophysiological effect of RhoA is to influence the disruption of the F-actin cytoskeleton by regulating NF-κB activities.

Analysis copy number variation of Chinese children in early-onset epileptic encephalopathies with unknown cause.

Copy number variations (CNVs) play an important role in the genetic etiology of unknown cause early‐onset epileptic encephalopathies (EOEEs), but the genomic CNVs analysis of Chinese EOEEs children was rare. Here, we identified CNVs by single nucleotide polymorphism array in 116 patients with different subtypes of EOEEs. Of 116 patients 17 (14.66%) carried 19 large CNVs. A total of 14 CNVs in 12 patients were further validated: four of the CNVs were classified as de novo, seven were maternal, and three were paternal. Follow‐up of those 12 patients showed that 5 had been seizure‐free for at least 9 months, 5 had seizures several times per month or per year, and 2 had seizures everyday. But eight patients have profound developmental delay. In this study, we found at least 3.4% of patients had pathogenic CNVs. For the patients, our study laid the foundation for prenatal interventions for their families. Further, we identified potential candidate gene involved in EOEEs. The association of CNVs and clinical features will contribute to the understanding of EOEEs.

Screening and identification of dynamin-1 interacting proteins in rat brain synaptosomes.

Dynamin-1 is a multi-domain GTPase that is crucial for the fission stage of synaptic vesicle recycling and vesicle trafficking. In this study, we constructed prokaryotic expression plasmids for the four functional domains of dynamin-1, which are pGEX-4T-2-PH, pGEX-4T-2-PRD, pGEX-4T-2-GED and pGEX-4T-2-GTPase. Glutathione S-transferase pull-down, co-immunoprecipitation (co-IP), and liquid chromatography/mass spectrometry were used to screen and identify dynamin-1 interacting proteins in rat brain synaptosomes. We identified a set of 63 candidate protein interactions, including 36 proteins interacting with dynamin-1 C-terminal proline-rich domain (PRD), 14 with pleckstrin-homology domain (PH), 7 with GTPase effector domain (GED) and 6 with GTPase domain, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. We selected three previously unreported dynamin-1 interacting proteins to verify their interaction with dynamin-1 under native conditions. Using co-IP, we found that Rab GDP-dissociation inhibitor (Rab GDI) and chloride channel 3 (ClC-3) do interact with dynamin-1, but not with TUC-4b (the TOAD-64/Ulip/CRMP (TUC) family member). Those novel interactions detected in our study offer valuable insight into the protein-protein interacting network that could enhance our understanding of dynamin-1 mediated synaptic vesicle recycling.

Measurement of Rho-kinase and CD4~+CD25~+ regulatory T cells in the peripheral blood in asthmatic patients

OBJECTIVE To determine the levels of Rho-kinase and CD4+CD25+ regulatory T cells in patients with asthma, and the relationship between Rho-kinase and CD4+CD25+ regulatory T cells. METHODS We included 16 patients with moderate to severe asthma in the research group and 14 healthy people as the control group. The levels of Rho-kinase in the 2 groups were measured by ELISA. The level of CD4+CD25+ regulatory T cells in the 2 groups was measured by flow cytometry. The pulmonary function was measured by spirometer. RESULTS The level of Rho-kinase in the research group was higher than that in the healthy controls (P<0.05). The level of CD4+CD25+ regulatory T cells in the healthy controls was higher than that of the research group (P<0.05). There was no correlation between the level of Rho-kinase in the peripheral blood of the 2 groups and forced expiratiory volume at the first second/ forced vital capacity (FEV1%) (r=-0.491, P>0.05). The level of CD4+CD25+ regulatory T cells in the peripheral blood of the 2 groups showed a positive correlation with FEV1% (r=0.380, P=0.038). There was no correlation between the level of Rho-kinase and the level of CD4+CD25+ regulatory T cells in the peripheral blood of the 2 groups (r=-0.438, P>0.05). CONCLUSION Rho-kinase and CD4+CD25+ regulatory T cells may play a key role in the pathogenesis of asthma.