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Pulmonary exposure to certain engineered nanomaterials (ENMs) causes chronic lesions like fibrosis and cancer in animal models as a result of unresolved inflammation. Resolution of inflammation involves the time-dependent biosynthesis of lipid mediators (LMs)-in particular, specialized pro-resolving mediators (SPMs). To understand how ENM-induced pulmonary inflammation is resolved, we analyzed the inflammatory and pro-resolving responses to fibrogenic multi-walled carbon nanotubes (MWCNTs, Mitsui-7) and low-toxicity fullerenes (fullerene C60, C60F). Pharyngeal aspiration of MWCNTs at 40 μg/mouse or C60F at a dose above 640 μg/mouse elicited pulmonary effects in B6C3F1 mice. Both ENMs stimulated acute inflammation, predominated by neutrophils, in the lung at day 1, which transitioned to histiocytic inflammation by day 7. By day 28, the lesion in MWCNT-exposed mice progressed to fibrotic granulomas, whereas it remained as alveolar histiocytosis in C60F-exposed mice. Flow cytometric profiling of whole lung lavage (WLL) cells revealed that neutrophil recruitment was the greatest at day 1 and declined to 36.6% of that level in MWCNT- and 16.8% in C60F-treated mice by day 7, and to basal levels by day 28, suggesting a rapid initiation phase and an extended resolution phase. Both ENMs induced high levels of proinflammatory leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) with peaks at day 1, and high levels of SPMs resolvin D1 (RvD1) and E1 (RvE1) with peaks at day 7. MWCNTs and C60F induced time-dependent polarization of M1 macrophages with a peak at day 1 and subsequently of M2 macrophages with a peak at day 7 in the lung, accompanied by elevated levels of type 1 or type 2 cytokines, respectively. M1 macrophages exhibited preferential induction of arachidonate 5-lipoxygenase activating protein (ALOX5AP), whereas M2 macrophages had a high level expression of arachidonate 15-lipoxygenase (ALOX15). Polarization of macrophages in vitro differentially induced ALOX5AP in M1 macrophages or ALOX15 in M2 macrophages resulting in increased preferential biosynthesis of proinflammatory LMs or SPMs. MWCNTs increased the M1- or M2-specific production of LMs accordingly. These findings support a mechanism by which persistent ENM-induced neutrophilic inflammation is actively resolved through time-dependent polarization of macrophages and enhanced biosynthesis of specialized LMs via distinct ALOX pathways.

An improved understanding of the molecular mechanisms in synapse formation provides insight into both learning and memory and the etiology of neurodegenerative disorders. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein methyltransferase that negatively regulates synaptic gene expression and inhibits neuronal differentiation. Despite its regulatory function in neurons, little is known about the CARM1 cellular location and its role in dendritic maturation and synapse formation. Here, we examined the effects of CARM1 inhibition on dendritic spine and synapse morphology in the rat hippocampus. CARM1 was localized in hippocampal post-synapses, with immunocytochemistry and electron microscopy revealing co-localization of CARM1 with post-synaptic density (PSD)-95 protein, a post-synaptic marker. Specific siRNA-mediated suppression of CARM1 expression resulted in precocious dendritic maturation, with increased spine width and density at sites along dendrites and induction of mushroom-type spines. These changes were accompanied by a striking increase in the cluster size and number of key synaptic proteins, including N-methyl-d-aspartate receptor subunit 2B (NR2B) and PSD-95. Similarly, pharmacological inhibition of CARM1 activity with the CARM1-specific inhibitor AMI-1 significantly increased spine width and mushroom-type spines and also increased the cluster size and number of NR2B and cluster size of PSD-95. These results suggest that CARM1 is a post-synaptic protein that plays roles in dendritic maturation and synaptic formation and that spatiotemporal regulation of CARM1 activity modulates neuronal connectivity and improves synaptic dysfunction.

Activity of neprilysin (NEP), the major protease which cleaves amyloid-β peptide (Aβ), is reportedly reduced in the brains of patients with Alzheimer’s disease (AD). Accumulation of Aβ generates reactive oxygen species (ROS) such as 4-hydroxynonenal (HNE), and then reduces activities of Aβ-degrading enzymes including NEP. Xanthorrhizol (Xan), a natural sesquiterpenoid, has been reported to possess antioxidant and anti-inflammatory properties. The present study examined the effects of Xan on HNE- or oligomeric Aβ42-induced oxidative modification of NEP protein. Xan was added to the HNE- or oligomeric Aβ42-treated SK-N-SH human neuroblastoma cells and then levels, oxidative modification and enzymatic activities of NEP protein were measured. Increased HNE levels on NEP proteins and reduced enzymatic activities of NEP were observed in the HNE- or oligomeric Aβ42-treated cells. Xan reduced HNE levels on NEP proteins and preserved enzymatic activities of NEP in HNE- or oligomeric Aβ42-treated cells. Xan reduced Aβ42 accumulation and protected neurones against oligomeric Aβ42-induced neurotoxicity through preservation of NEP activities. These findings indicate that Xan possesses therapeutic potential for the treatment of neurodegenerative diseases, including AD, and suggest a potential mechanism for the neuroprotective effects of antioxidants for the prevention of AD.

Autophagy is a cellular degradation process for cellular aggregates and unneeded cellular compartments including damaged mitochondria, ER, and peroxisomes. Melanosome is cellular organelle that is the cellular site of generation, storage and transports of melanin in melanocytes. Despite potential importance of autophagy, the role of autophagy in melanogenesis and melanosome autophagy are largely unknown. In here, we identified 3′-hydroxydaidzein (3′-ODI) as an autophagy inducer from a phytochemical library screening. Treatment with 3′-ODI significantly reduced α-MSH-mediated melanogenesis but efficiently increased autophagy both in melanoma cells and melanocytes. Furthermore, inhibition of autophagy significantly reduced the anti-melanogenic effects of 3′-ODI in α-MSH-stimulated melanoma cells. Taken together, these results suggest that autophagy mediates anti-melanogenic activity of 3′-ODI.

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3beta (GSK-3beta) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGF-induced phosphorylation of Akt and GSK-3beta, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3beta, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3beta pathway.

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.

SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck. It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 ProteinInteracting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

The cellular localization of alpha3beta1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of alpha3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and alpha3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. Alpha-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the alpha3A subunit and other myofibrillar proteins into sarcomeres revealed that alpha3A was integrated into sarcomeres following incorporation of alpha-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that alpha3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that alpha3A integrins participate in the initial assembly of myofibrillar proteins. The alpha3B, beta1A and beta1D subunits were also localized in costameres, where they formed alpha3Abeta1A, alpha3Abeta1D and alpha3Bbeta1A heterodimers. The alpha3Bbeta1D heterodimer, however, was not found in cardiac myocytes. The antisera raised against the cytoplasmic domains of alpha3A, alpha3B, beta1A and beta1D caused disruption of sarcomere structure. Thus, the myofibril-extracellular matrix linkages mediated by isoforms of alpha3beta1 integrin may play a crucial role in the stabilization of myofibril assembly and in the maintenance of sarcomere structure. Co-immunoprecipitation experiments revealed that beta1A, but not beta1D, interacts with the Nck signaling protein, suggesting that Nck participates in downstream signaling triggered by beta1A and that the beta1A-mediated signaling pathway is distinct from that of beta1D.

Epidemiological studies suggest that the treatments of anti-inflammatory agents and anti-oxidants slow the progress of neurological diseases. Lignans are anti-oxidants and phytoestrogens found in a variety of plants. In this study, we investigated the neuroprotective effect of macelignan on glutamate-induced neurotoxicity and reactive oxygen species (ROS) in murine hippocampal HT22 cell line. Macelignan significantly attenuated the ROS production and neurotoxicity induced by glutamate in HT22 cell. Also, the properties of macelignan as an anti-inflammatory agent were investigated in microglials activation by lipopolysaccharide (LPS). It potently suppressed the expression of cyclooxygenase-2 and inducible nitric oxide synthase, that consequently resulted in the reduction of nitric oxide in LPS-treated microglial cells. It also significantly suppressed the production of pro-inflammatory cytokine tumor necrosis factor-alpha and interleukin-6. These results suggest that macelignan possesses therapeutic potentials against neurodegenerative diseases with oxidative stress and neuroinflammation.

Amyloid-beta (Aβ) peptide accumulation in the brain is a pathological hallmark of all forms of Alzheimer's disease. An imbalance between Aβ production and clearance from the brain may contribute to accumulation of neurotoxic Aβ and subsequent synaptic loss, which is the strongest correlate of the extent of memory loss in AD. The activity of neprilysin (NEP), a potent Aβ-degrading enzyme, is decreased in the AD brain. Expression of HuD, an mRNA-binding protein important for synaptogenesis and neuronal plasticity, is also decreased in the AD brain. HuD is regulated by protein kinase Cε (PKCε), and we previously demonstrated that PKCε activation decreases Aβ levels. We hypothesized that PKCε acts through HuD to stabilize NEP mRNA, modulate its localization, and support NEP activity. Conversely, loss of PKCε-activated HuD in AD leads to decreased NEP activity and accumulation of Aβ. Here we show that HuD is associated with NEP mRNA in cultures of human SK-N-SH cells. Treatment with bryostatin, a PKCε-selective activator, enhanced NEP association with HuD and increased NEP mRNA stability. Activation of PKCε also increased NEP protein levels, increased NEP phosphorylation, and induced cell surface expression. In addition, specific PKCε activation directly stimulated NEP activity, leading to degradation of a monomeric form of Aβ peptide and decreased Aβ neuronal toxicity, as measured by cell viability. Bryostatin treatment also rescued Aβ-mediated inhibition of HuD-NEP mRNA binding, NEP protein expression, and NEP cell membrane translocation. These results suggest that PKCε activation reduces Aβ by up-regulating, via the mRNA-binding protein HuD, Aβ-degrading enzymes such as NEP. Thus, PKCε activation may have therapeutic efficacy for AD by reducing neurotoxic Aβ accumulation as well as having direct anti-apoptotic and synaptogenic effects.