Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

No abstract

In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC50 of 160 nM, while the GST-Moj-NN had an IC50 of 493 nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC50 of 11 nM, followed by the GST-Moj-NN (IC50 of 28 nM), and the GST-Moj-DM (IC50 of 46 nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5 μM GST-Moj peptides for 24 h. At 5 μM GST-Moj-DM peptide, 13.56% +/- 2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express αvβ3, αv, α6, β1, and β3 integrin receptors.

Increased retinal vascular permeability contributes to macular edema, a leading cause of vision loss in eye pathologies such as diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusions. Pathological changes in vascular permeability are driven by growth factors such as VEGF and pro-inflammatory cytokines such as TNF-α. Identifying the pro-barrier mechanisms that block vascular permeability and restore the blood-retinal barrier (BRB) may lead to new therapies. The cAMP-dependent guanine nucleotide exchange factor (EPAC) exchange-protein directly activated by cAMP promotes exchange of GTP in the small GTPase Rap1. Rap1 enhances barrier properties in human umbilical endothelial cells by promoting adherens junction assembly. We hypothesized that the EPAC-Rap1 signaling pathway may regulate the tight junction complex of the BRB and may restore barrier properties after cytokine-induced permeability. Here, we show that stimulating EPAC or Rap1 activation can prevent or reverse VEGF- or TNF-α-induced permeability in cell culture and Moreover, EPAC activation inhibited VEGF receptor (VEGFR) signaling through the Ras/MEK/ERK pathway. We also found that Rap1B knockdown or an EPAC antagonist increases endothelial permeability and that VEGF has no additive effect, suggesting a common pathway. Furthermore, GTP-bound Rap1 promoted tight junction assembly, and loss of Rap1B led to loss of junctional border organization. Collectively, our results indicate that the EPAC-Rap1 pathway helps maintain basal barrier properties in the retinal vascular endothelium and activation of the EPAC-Rap1 pathway may therefore represent a potential therapeutic strategy to restore the BRB.

Maintenance and regulation of the vascular endothelial cell junctional complex is critical for proper barrier function of the blood-brain barrier (BBB) and the highly related blood-retinal barrier (BRB) that help maintain proper neuronal environment. Recent research has demonstrated that the junctional complex is actively maintained and can be dynamically regulated. Studies focusing on the mechanisms of barrier formation, maintenance, and barrier disruption have been of interest to understanding development of the BBB and BRB and identifying a means for therapeutic intervention for diseases ranging from brain tumors and dementia to blinding eye diseases. Research has increasingly revealed that small GTPases play a critical role in both barrier formation and disruption mechanisms. This review will summarize the current data on small GTPases in barrier regulation with an emphasis on the EPAC-Rap1 signaling pathway to Rho in endothelial barriers, as well as explore its potential involvement in paracellular flux and transcytosis regulation.

No abstract

Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin-V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68% +/− 3.09 of treated HUVEC, and 35.86% +/− 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

No abstract