Membrane-Cytoskeleton Connectivity in Alzheimer’s Disease:From Aβ Clearance to TauSpreading by Microglia

Alzheimer’s disease (AD) is a progressive irreversible neurodegenerative disease affecting about 5.8 million Americans. There’s no cure for AD yet. Amyloid plaques and neurofibrillary tangles are two hallmarks of AD. And their major component, Amyloid-beta (Aβ) and tau protein may be responsible for the cause and progression of AD. Microglia is a type of glial cell important for maintaining brain homeostasis. Cytosolic phospholipase A2 (cPLA2) cleaves phospholipid, the main component of cellular membrane, and release signaling molecules. In this study, we explored the impact of cPLA2 on membrane physical properties such as membrane cytoskeleton connectivity and related signaling pathways in microglia. Effects of cPLA2 on oligomeric Aβ clearance and Tau propagation in AD were also investigated. Our study showed that inhibition of cPLA2 activity increased the membrane cytoskeleton connectivity leading to attenuated oligomeric Aβ uptake by microglia. The results also suggested that inhibition of cPLA2 with arachidonyl trifluoromethyl ketone increased the production of exosomes secreted by microglia fed with Tau. Detailed mechanism on how ATK and cPLA2 affect the production of exosome in microglia and spreading of Tau through exosome will be explored with future experiments. Together, this study provided insight for Aβ clearance and Tau spreading in AD by altering cPLA2 activity in microglia and may be helpful for the development of AD treatments

Additional file 2: of Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation

Excel S2. The enriched DEGs GO terms between control and iron-limitation groups using bioinformatics methods. (XLSX 42 kb

Results of the artificial infection experiment using by the isolate strain GYX2014-1.

<p>Results of the artificial infection experiment using by the isolate strain GYX2014-1.</p

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Drug sensitivity test results of the isolated strain GYX2014-1.

<p>Drug sensitivity test results of the isolated strain GYX2014-1.</p

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK

<div><p>Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.</p></div

Additional file 3: of Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation

Excel S3. The enriched DEPs GO terms between control and iron-limitation groups using bioinformatics methods. (XLSX 42 kb

Schematic description of transendothelial permeability assays.

<p>(A) Diagram depicting method for Dextran assay protocol. (B) Diagram depicting the measurement using the TEER protocol.</p

Effects of TNFα, IL-1β and LPS on morphology of hCMEC/D3 cells.

<p>(A) Cells were treated with or without TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL) for 24 hours and observed under bright field microscope as described in text. Representative pictures were taken from different areas in the field. (B) Ten cells from each picture were randomly selected for measurement using the Image J protocol. Results are mean ± SD from 3 experiments. One-way ANOVA with Bonferroni post-test showed a significant difference (p<0.01) between control and TNFα. (C) Representative bright field photomicroscope pictures to assess effects of MEK1/2 and p-p38MAPK inhibitors on TNFα-induced morphological changes. Cells were pretreated with U0126 (2 μM) and SB202190 (2 μM) for 15 min prior to treatment with TNFα (10 ng/mL) for 24 h. (D) Results are analyzed as in (B). Data are expressed as the mean ± SD of three experiments. Two-way ANOVA showed a significant main effect of TNFα (p = 0.0013). The effects of the inhibitors were not significant.</p

Effects of TNFα, IL-1β and LPS on p-ERK1/2 and p-p38MAPK expression in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (A, B, C), IL-1β (D) and LPS (E) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and phospho-ERK1/2 (P- ERK), total ERK1/2 (T-ERK), phospho-p38MAPK (P-P38), total p38MAPK (T-P38) and β-actin expression pattern were analyzed by immunoblotting assay. Quantification of phospho-proteins was determined through assay of PIphospho/PItotal/PIβ-actin and then normalized to control. Results of (B) and (C) are mean ± SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests **P<0.01, ***P<0.001 compared with no treatment control). Results of (D) and (E) are representation of two repeated experiments.</p