Journal: Cell & Bioscience

Article Title: Salidroside reduces neuropathology in Alzheimer’s disease models by targeting NRF2/SIRT3 pathway

doi: 10.1186/s13578-022-00918-z

Figure Lengend Snippet: SAL ameliorates Aβ-induced neurite and mitochondrial damage. A Schematic overview shows the workflow of SH-SY5Y differentiation and primary neuronal culture for SAL efficacy study by assessing neurite morphology and mitochondrial dynamics. B Immunofluorescence (IF) of neuronal marker Tuj1 (green) in differentiated SH-SY5Y cells, treated with indicated Aβ42 oligomers or SAL (see “ ”). White rectangles indicate amplified images at lower panels. Scale bar, 50 μm. C As in B , except primary neurons used. D Quantification of neurite length of B and C . The average neurite length without additional treatment was regarded as control (CTL). In each condition, at least 7 random images (each image includes ≥ 20 cells) from 3 independent experiments were used for quantification. E IF of TOM20 shows the mitochondrial segments in differentiated neurites of SH-SY5Y cells with indicated treatments. Scale bar, 10 μm. The quantification shows the length of mitochondrial (mito.) segments (n ≥ 400) in F , and the mitochondrial density indicating by the ratio of mito. length to occupied neurite (shaft) length (n ≥ 20) in G . Error bars indicate the mean ± SD from at least 20 neurites of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; one-way ANOVA test

Article Snippet: Aβ42 peptide was purchased from GenScript.

Techniques: Immunofluorescence, Marker, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Preliminary Study on Clusterin Protein (sCLU) Expression in PC-12 Cells Overexpressing Wild-Type and Mutated (Swedish) AβPP genes Affected by Non-Steroid Isoprenoids and Water-Soluble Cholesterol

doi: 10.3390/ijms20061481

Figure Lengend Snippet: Effect of mevalonate (MEV, 100 μM), farnesol (FOH, 10 μM), geranylgeraniol (GGOH, 10 μM) or water soluble cholesterol (Chol-PEG, 1 mM) on selected protein expressions in cells affected by MEV pathway modulators ( A for atorvastatin – ATR, B for simvastatin – SIM). Western blot analysis of proteins: amyloid precursor protein (AβPP, 110 kDa), soluble amyloid protein precursor alpha (sAβPPα, 100 kDa), clusterin (CLU, 60 and 40 kDa), amyloid-β 1-16 (Aβ, 4 kDa), actin (42 kDa) as house-keeping protein in empty vector (G), AβPP wild type gene ( AβPP -wt, W), and AβPP Swedish mutations gene ( AβPP -sw, S) transfected cells. The results are indicative of three independent experiments with similar results.

Article Snippet: After transfer to 0.2 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad) membranes were blocked in either 5% ( w / v ) nonfat dried milk or 1% ( w / v ) BSA (Sigma Aldrich) diluted in Tris-buffered saline containing 0.1% ( v / v ) Tween 20 (TBS-T), and probed overnight at 4 °C with the respective primary antibodies, as follows: actin, clusterin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), AβPP, sAβPPα, beta amyloid 1–16 (6E10) (Covance, Inc., Princeton, NY, USA), AβPP (Merck Millipore, Darmstadt, Germany).

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: PA ameliorates Aβ42 oligomer‐induced learning and memory deficits in mice. (A) The spontaneous alternation rate in Y‐maze test. (B) Distance and (C) escape latency to the target platform on the learning phase of the Morris water maze assessment. (D) Time spent on the target quadrant during the spatial probe phase. (E) A number of cross the platform during the spatial probe trial. (F) Representative tracing recordings for the spatial probe trial (each group n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001; distance traveled to target was analyzed using two‐way analysis of variance (ANOVA), and the others using one‐way ANOVA with Tukey's post‐hoc test. Bars represent mean ± SD.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: Inhibitory effects of PA on inflammation induced by Aβ42 oligomer in mice. (A) TNF‐α, (B) IL‐1β, and (C) IL‐6 were examined using ELISA ( n = 6 per group). (D) Immunofluorescence analysis of Iba‐1, bar = 25 μm. (E) Western blots and (F) quantitative analysis of Iba‐1 ( n = 3 per group). All values are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the AD model group.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: Effect of PA and Aβ42 treatment on the cell viability of BV2 and N2a cells. (A) Cell viability of BV2 cells and (B) N2a cells after 24 h treatment of PA. (C) Cell viability of BV2 cells after 24 h treatment of Aβ42. (D) Cell viability of BV2 cells after 2 h pretreatment with PA followed by 24 h treatment with PA and Aβ42. (E) Cell viability of N2a cells after cultured under conditional medium from BV2. (F–I) Apoptosis of N2a was analyzed by flow cytometry under conditional medium from BV2. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the Aβ42 stimulated group. One‐way ANOVA with Tukey's post‐hoc test. Bars represent mean ± SD.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques: Cell Culture, Flow Cytometry

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: Western blot analysis for PPAR γ/NF‐κB pathway‐related proteins in mice. (A) Western blot results. (B–D) Quantitative analysis of corresponding proteins ( n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the Aβ42‐stimulated group. There were eight mice in each group; one‐way ANOVA with Tukey's post‐hoc test. Bars represent mean ± SD.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques: Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: PPARγ antagonists reversed the effects of PA on the PPARγ/NF‐κB pathway and inflammation factors in BV2 microglia. (A–C) The secretion of inflammatory cytokines in BV2 cells after the treatment of PA; (D–F) The secretion of inflammatory cytokines in BV2 cells and after pretreatment with PPARγ inhibitors. (G) Western blot analysis of the PPARγ/NF‐κB pathway and (H–J) quantitative analysis of corresponding proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the Aβ42 stimulated group; & p < 0.05, && p < 0.01, &&& p < 0.001, and &&&& p < 0.001 compared with the PA treatment group. One‐way ANOVA with Tukey's post‐hoc test. n = 6 per group for ELISA analysis, and n = 3 for Western analysis. Bars represent mean ± SD.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: NF‐κB expressions in the nucleus and cytoplasm after adding the inhibitor of PPARγ. (A) Western blots analysis for NF‐κB p65 subunit in nuclear and cytosolic and quantitative analysis of corresponding proteins. (B) The representative immunofluorescence images showing translocation of NF‐κB (p65, red) to the nucleus (blue) after treatment with Aβ42 oligomers or pretreat with PA or adding the inhibitor of GW9662. Bar = 50 μm. (C) Percent of nuclear NF‐κB p65‐positive cells. Values were expressed as the mean of at least three independent replicates, whose means resulted from 50 cells from three independent fields of view. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the Aβ42 stimulated group; & p < 0.05, && p < 0.01, &&& p < 0.001, and &&&& p < 0.001 compared with the PA treatment group. n = 3 for Western analysis, one‐way ANOVA with Tukey's post‐hoc test. Bars represent mean ± SD.

Article Snippet: Aβ42 oligomer powder was obtained from ChinaPeptides Co., Ltd. (Suzhou, China).

Techniques: Western Blot, Immunofluorescence, Translocation Assay

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: (A) Experimental scheme: Four-month-old wild-type female mice (B6SJLF1) were subjected to a pre-infusion of PBS or TNF-alpha via the left internal carotid artery over a duration of 15 minutes. Following the infusion, a waiting period of 60 minutes was observed before administering a bolus injection of 125 I-Aβ42 (150 μCi) into the femoral vein. Blood samples were collected periodically from the femoral artery at intervals ranging from 0 to 40 minutes. These blood samples were subsequently centrifuged to isolate the plasma, and intact 125 I-Aβ42 was precipitated using TCA. Radioactivity levels were quantified utilizing a gamma counter. After the final blood sampling event, the mice underwent transcardial perfusion with an excess of PBS, followed by dissection of brain regions. The level of 125 I-Aβ42 activity within the brain was then assessed using a gamma counter. (B) Plasma concentration versus time profile of 125 I-Aβ42 in mice infused with TNF alpha or PBS control. The observed values are presented alongside the predicted curves (C) The permeability-surface area (PS) products for 125 I-Aβ42 for each brain region were estimated in mice infused with TNF alpha or PBS control. Data are expressed as mean ± standard deviation. Significance levels: *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni’s post-tests. (D) Bar graph depicts the percentage of the injected dose of 125 I-Aβ42 in the whole brain per gram of tissue in TNF alpha-infused mice compared to PBS-infused mice Data are expressed as mean ± standard deviation. Significance levels: *p<0.05; determined by two-tailed t-test. (E) The brain influx (Ki) of 125 I-Aβ42 was estimated by the slope obtained from Gjedde-Patlak graphical analysis. Inset table values are mean ± SD (n = 4). Significance levels: *p < 0.05; two-tailed t-test.

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Injection, Radioactivity, Sampling, Dissection, Activity Assay, Concentration Assay, Control, Permeability, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: (A) Experimental design. PBMECs or hCMEC/D3 cultured in 6 well plates were treated with TNF-alpha PBMECs (10 ng/mL), hCMEC/D3 (20 ng/mL) for 24 hrs. in low serum medium at 37°C. Subsequently, the cells were treated with FITC-Aβ42 (1 μM) and incubated for 1 hr. at 37°C. Intracellular fluorescence of samples was assessed by flow cytometry. (B&C). Bar charts display the intracellular median fluorescence intensity (MFI) of (B) PBMECs or (C) hCMEC/D3 incubated with FITC-Aβ42 in TNF-alpha treated and control groups. Data are expressed as mean ± S.D. Significance levels: *p<0.05,**p<0.01; determined by two-tailed t-test. (D) Confocal micrographs illustrate increased FITC-Aβ42 uptake in hCMEC/D3 cells treated with TNF-alpha compared to control cells. FITC-Aβ42 is visualized in green, while the nuclei are stained blue (DAPI). Scale bar = 20 μm

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Cell Culture, Incubation, Fluorescence, Flow Cytometry, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: (A) Experiment design. hCMEC/D3 were transfected with or without cofilin siRNA (25 nM) for 6 hours. The treatment media was removed and replaced with blank D3 media for 24 hours at 37°C. Subsequently, the cells were treated with TNF alpha (20 ng/mL) in low serum (1%) medium at 37°C. The cells were then treated with FITC-Aβ42 (1 μM) and incubated for 1 hr. at 37°C. Intracellular fluorescence of samples was assessed by flow cytometry and protein expression was determined by western blot. (B&C). Histogram and bar chart depicting intracellular median fluorescence intensity (MFI) of hCMEC/D3 incubated with or without cofilin siRNA, in the presence of FITC-Aβ42 and TNF-alpha. Data expressed as mean ± S.D. Statistical significance: *p<0.05; determined by two-tailed t-test. (D&E) Bar chart demonstrates quantification of immunoblots by densitometry for phospho-cofilin, both normalized to GAPDH protein levels. Data expressed as mean ± S.D. Statistical significance: ****p<0.0001; determined by two-tailed t-test.

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Transfection, Incubation, Fluorescence, Flow Cytometry, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: (A) Experimental scheme. hCMEC/D3 monolayers were pre-treated with dynasore (80 μM, 2hrs, 37 °C.) in low serum medium, followed by spiking the cells with TNF alpha (20 ng/mL) and incubating them for 22 hours at 37°C. Subsequently, the cells were treated with 125 I-Aβ42 (5 μCi) or FITC-Aβ42 (1 μM) and incubated for 1 hr. at 37°C. Samples were assessed by measuring 125 I activity using a gamma counter or by live cell imaging using confocal microscopy. ( B) Bar chart demonstrates dynasore-mediated reduction in 125 I-Aβ42 counts per minute (cpm) following treatment with TNF alpha. Data expressed as mean ± S.D. Statistical significance: *p<0.01, *p<0.05; determined by one-way ANOVA. ( C) Dynasore inhibits TNF alpha induced increase in FITC-Aβ42 (green) endothelial cellular accumulation, as depicted in the Z-stack confocal micrographs. Scale bar (20 μm).

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Incubation, Activity Assay, Live Cell Imaging, Confocal Microscopy

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: TNF alpha induces an increase in cofilin phosphorylation at the serine 3 site, facilitating actin cytoskeleton remodeling and actin filament polymerization. This process promotes endothelial membrane invagination, facilitating Aβ42 uptake by BBB endothelial cells. Additionally, TNF alpha enhances dynamin phosphorylation, which promotes membrane scission and endocytic vesicle release containing Aβ42.

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Membrane

Journal: bioRxiv

Article Title: Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

doi: 10.1101/2025.01.28.635286

Figure Lengend Snippet: (A) Experimental scheme: hCMEC/D3 monolayers cultured on 35 mm glass coverslip round bottom dishes were treated with TNF alpha (20 ng/mL) 24 hours in low serum medium at 37°C. Subsequently, the cells were incubated with FITC-Aβ42 (1 μM) for 30 minutes at 37°C, followed by co-incubation with LysoTracker red (75 nM) for another 30 minutes at 37°C. The treatment medium was then replaced with low serum D3 media (1% FBS), and live cell imaging was conducted using a confocal microscope. (B) Confocal micrographs illustrate the absence of colocalization between lysosomes and FITC-Aβ42 in the presence of TNF alpha. FITC-Aβ42 is visualized in green, while lysosomes are stained red. Scale bar = 20 μm

Article Snippet: The Aβ42 peptides (Aapptec, Louisville, KY) were labeled with the 125 I radionuclide (PerkinElmer Life and Analytical Sciences, Boston, MA) utilizing the chloramine-T procedure as previously described( ; ).

Techniques: Cell Culture, Incubation, Live Cell Imaging, Microscopy, Staining