Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.</p> <p><b>Methods</b></p> <p>We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).</p> <p><b>Results</b></p> <p>METH induced microglial cell death in a concentration-dependent manner (EC₅₀ = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.</p> <p><b>Conclusions</b></p> <p>These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.</p

Plasma surface modification of two-component composite scaffolds consisting of 3D-printed and electrospun fiber components from biodegradable PLGA and PLCL

In this study, two-component, morphologically composite scaffolds consisting of a 3D-printed component and an electrospun fiber component were fabricated and treated with a nitrogen-argon (N2-Ar) plasma to enhance their surface properties. The 3D-printed component provided mechanical strength, while the electrospun fibrous component acted as a mimic to the extracellular matrix to improve cell-substrate interactions. Two biodegradable polyesters, poly(L-lactide-co-ε-caprolactone) (PLCL) and poly(L-lactide-co-glycolide) (PLGA), were used to create the scaffolds. The resulting 3D/E/N2-Ar scaffolds were characterized in terms of surface properties (morphology, chemical compositions, wettability, roughness, crystallinity), degradation, mechanical properties, and cell cytotoxicity, cell attachment and proliferation, LDH release and cell apoptosis. Results showed that the plasma treatment significantly increased the surface roughness, wettability, and hydrophilicity of the scaffolds. The 3D-printed component provided sufficient mechanical support, while the electrospun fiber component promoted cell attachment and proliferation. Following plasma treatment, the water contact angle of the scaffolds was greatly reduced from 124.0 ± 1.8° (PLCL) and 119.6 ± 1.4° (PLGA), to 0° and persisted even after 168 days. Human Schwann cells (SCs) showed excellent viability on both 3D/E/N2-Ar and 3D/E scaffolds were in excess of 95%. Cells cultivated on the 3D/E/N2-Ar scaffolds, with higher surface roughness, displayed significant increase in attachment and proliferation and a higher presence of healthy cells when compared with untreated 3D/E scaffolds. Both PLCL and PLGA scaffolds showed potential for use in biomedical applications. Although PLGA performed slightly better in terms of cell behavior, PLCL exhibited a slower degradation rate and higher tensile strain. These results demonstrate the potential of these designed scaffolds to support cell regeneration in clinically relevant devices such as nerve guide conduits and nerve protectant wraps

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins