Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress.

BACKGROUND: Maternal exposure to environmental stressors poses a risk to fetal development. Oxidative stress (OS), microglia activation, and inflammation are three tightly linked mechanisms that emerge as a causal factor of neurodevelopmental anomalies associated with prenatal ethanol exposure. Antioxidants such as glutathione (GSH) and CuZnSOD are perturbed, and their manipulation provides evidence for neuroprotection. However, the cellular and molecular effects of GSH alteration in utero on fetal microglia activation and inflammation remain elusive. METHODS: Ethanol (EtOH) (2.5 g/kg) was administered to pregnant mice at gestational days 16-17. One hour prior to ethanol treatment, N-acetylcysteine (NAC) and L-buthionine sulfoximine (BSO) were administered to modulate glutathione (GSH) content in fetal and maternal brain. Twenty-four hours following ethanol exposure, GSH content and OS in brain tissues were analyzed. Cytokines and chemokines were selected based on their association with distinctive microglia phenotype M1-like (IL-1ß, IFN γ, IL-6, CCL3, CCL4, CCL-7, CCL9,) or M2-like (TGF-ß, IL-4, IL-10, CCL2, CCL22, CXCL10, Arg1, Chi1, CCR2 and CXCR2) and measured in the brain by qRT-PCR and ELISA. In addition, Western blot and confocal microscopy techniques in conjunction with EOC13.31 cells exposed to similar ethanol-induced oxidative stress and redox conditions were used to determine the underlying mechanism of microglia activation associated with the observed phenotypic changes. RESULTS: We show that a single episode of mild to moderate OS in the last trimester of gestation causes GSH depletion, increased protein and lipid peroxidation and inflammatory responses inclined towards a M1-like microglial phenotype (IL-1ß, IFN-γ) in fetal brain tissue observed at 6-24 h post exposure. Maternal brain is resistant to many of these marked changes. Using EOC 13.31 cells, we show that GSH homeostasis in microglia is crucial to restore its anti-inflammatory state and modulate inflammation. Microglia under oxidative stress maintain a predominantly M1 activation state. Additionally, GSH depletion prevents the appearance of the M2-like phenotype, while enhancing morphological changes associated with a M1-like phenotype. This observation is also validated by an increased expression of inflammatory signatures (IL-1ß, IFN-γ, IL-6, CCL9, CXCR2). In contrast, conserving intracellular GSH concentrations eliminates OS which precludes the nuclear translocation and more importantly the phosphorylation of the NFkB p105 subunit. These cells show significantly more pronounced elongations, ramifications, and the enhanced expression of M2-like microglial phenotype markers (IL-10, IL-4, TGF-ß, CXCL10, CCL22, Chi, Arg, and CCR2). CONCLUSIONS: Taken together, our data show that maintaining GSH homeostasis is not only important for quenching OS in the developing fetal brain, but equally critical to enhance M2 like microglia phenotype, thus suppressing inflammatory responses elicited by environmental stressors.

Imaging, Spectroscopic, Mechanical and Biocompatibility Studies of Electrospun Tecoflex® EG 80A Nanofibers and Composites Thereof Containing Multiwalled Carbon Nanotubes.

The present study discusses the design, development and characterization of electrospun Tecoflex® EG 80A class of polyurethane nanofibers and the incorporation of multiwalled carbon nanotubes (MWCNTs) to these materials. Scanning electron microscopy results confirmed the presence of polymer nanofibers, which showed a decrease in fiber diameter at 0.5% wt. and 1% wt. MWCNTs loadings, while transmission electron microscopy showed evidence of the MWCNTs embedded within the polymer matrix. The fourier transform infrared spectroscopy and Raman spectroscopy were used to elucidate the polymer-MWCNTs intermolecular interactions, indicating that the C-N and N-H bonds in polyurethanes are responsible for the interactions with MWCNTs. Furthermore, tensile testing indicated an increase in the Young's modulus of the nanofibers as the MWCNTs concentration was increased. Finally, NIH 3T3 fibroblasts were seeded on the obtained nanofibers, demonstrating cell biocompatibility and proliferation. Therefore, the results indicate the successful formation of polyurethane nanofibers with enhanced mechanical properties, and demonstrate their biocompatibility, suggesting their potential application in biomedical areas.

DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells.

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

Inhibition of neddylation represses lipopolysaccharide-induced proinflammatory cytokine production in macrophage cells.

Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924 with a known anti-inflammatory agent (dexamethasone), on certain proinflammatory cytokines (TNF-α and IL-6) and the NF-κB signaling pathway in LPS-stimulated macrophages. We also used siRNA to block neddylation to assess the role of this molecular process during LPS-induced cytokine responsiveness. Our results demonstrate that blocking neddylation, either pharmacologically or using siRNA, abrogates the increase in certain proinflammatory cytokines secreted from macrophages in response to LPS. In addition, we have shown that MLN4924 and dexamethasone inhibit LPS-induced cytokine up-regulation at the transcriptional level, albeit through different molecular mechanisms. Thus, neddylation represents a novel molecular process in macrophages that can be targeted to prevent and/or treat the LPS-induced up-regulation of proinflammatory cytokines and the disease processes associated with their up-regulation.

Chemokines in the MPTP model of Parkinson's disease: absence of CCL2 and its receptor CCR2 does not protect against striatal neurodegeneration.

Recent studies have invoked inflammation as a major contributor to the pathogenesis of Parkinson's disease (PD). We determined the role of members of the chemokine system, key inflammatory mediators, in PD pathogenesis. In the MPTP model of murine PD, several chemokines, including CC chemokine ligand 2 (CCL2, Monocyte Chemoattractant Protein-1) and CCL3 (Macrophage Inflammatory Protein-1alpha), were upregulated in the striatum and the ventral midbrain. Astrocytes were the predominant source of CCL2 and CCL3 in the striatum and the substantia nigra, and dopaminergic neurons in the substantia nigra constitutively expressed these two chemokines. MPTP treatment resulted in decreased CCL2 expression and increased CCL3 expression in the surviving dopaminergic neurons. Because we found that CCL2 induced production of TNF-alpha in microglial cells, a cytokine known to play a detrimental role in PD, we anticipated that deletion of the genes encoding CCL2 and CCR2, its major receptor, would confer a protective phenotype. However, MPTP-induced striatal dopamine depletion was comparable in double knockout and wild-type mice. Our results demonstrate that chemokines such as CCL2 are induced following MPTP treatment, but that at least within the context of this PD model, the absence of CCL2 and CCR2 does not protect against striatal dopamine loss.