Journal: International Journal of Molecular Sciences

Article Title: Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer’s Disease (Mouse Model) through Autophagy Induced by Activating Axl

doi: 10.3390/ijms22115559

Figure Lengend Snippet: GAA facilitates Aβ42 degradation in microglial cells. ( A ) BV2 cells were treated with Rog (10 µM), GLT, and GLP at the indicated concentrations in the presence of Aβ42 (2 µM) for 24 h, and intracellular Aβ42 levels were measured using ELISA. Rog vs. Control: p = 0.0016; GLT-10 vs. Control: p =0.0005; GLT-30 vs. Control: p = 0.001; n = 3. ( B ) Aβ42 uptake by BV2 was assessed by applying FITC-labeled Aβ42 (1 µM) to BV2 cells in the presence of Rog (10 µM) and GLT at the indicated concentrations for 24 h. Accumulation of the fluorophore was analyzed by flow cytometry. n = 3. ( C – E ) BV2 cells were treated with Rog (10 µM), GAA, GAD, and GAG, respectively, at the indicated concentrations in the presence of Aβ42 (2 µM) for 24 h, and intracellular Aβ42 levels were measured using ELISA. ( C ) Rog vs. Control: p = 0.0002; GAA-5 vs. Control: p = 0.0132; GAA-20 vs. Control: 0.0002; n = 3. ( D ) Rog vs. Control: p = 0.0021; n = 3. ( E ) Rog vs. Control: p = 0.0009; n = 3. ( F ) Uptake of Aβ42 by BV2 was assessed by applying FITC-labeled Aβ42 (1 µM) to BV2 cells in the presence of Rog (10 µM) and GAA at the indicated concentrations for 24 h, and accumulation of the fluorophore was analyzed by fluorescence signal 1-height (FL1-H) of flow cytometry. Results were normalized to total cellular protein level, and DMSO at 0.1% was used as the control. n = 3. n is the number of replicates (biological and technical) used for each of the described results. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. indicated control.

Article Snippet: The human Aβ42 enzyme-linked immunosorbent assay (ELISA) kit was purchase from Cusabio (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Labeling, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer’s Disease (Mouse Model) through Autophagy Induced by Activating Axl

doi: 10.3390/ijms22115559

Figure Lengend Snippet: GAA promotes Aβ42 elimination through the autophagy pathway in microglial cells. ( A ) BV2 cells were treated with GAA (20 µM), the indicated antagonists of ADEs (arachidonic acid at 80 µM, sacubitrilat at 50 µM, enalapril maleate at 50 µM), autophagosomes (EACC at 2 µM) and lysosomes (chloroquine at 20 µM), in the presence of Aβ42 (2 µM) for 24 h, and intracellular Aβ42 levels were measured by ELISA. GAA + EACC vs. GAA: p = 0.0122; GAA + Chl vs. GAA: p = 0.0075; n = 3. ( B ) BV2 cells were treated with GAA (20 µM) for the indicated periods of time, and whole cell proteins were collected for western blot. 6 h vs. 0 h: p = 0.0219; 12 h vs. 0 h: p = 0.0467; n = 3. ( C – E ) BV2 cells were treated with GAA (20 µM) for the indicated times, and expression of Atg5, Becn1 and Map1lc3b was analyzed by qRT-PCR. ( C ) 6 h vs. 0 h: p = 0.0075; 12 h vs. 0 h: p = 0.0002; 24 h vs. 0 h: p = 0.0069; n = 3. ( D ) 3 h vs. 0 h: p = 0.0015; 6 h vs. 0 h: p = 0.0007; 12 h vs. 0 h: p = 0.0006; 24 h vs. 0 h: p = 0.0017; n = 3. ( E ) 3 h vs. 0: p = 0.0086; 6 h vs. 0 h: p = 0.0001; 12 h vs. 0 h: p < 0.0001; 24 h vs. 0 h: p = 0.0286; n = 3. ( F ) BV2 cells were treated with GAA (20 µM), the indicated agonist of AKT/PI3K (recilisib at 20 µM) and EACC (2 µM) for 6 h, and whole cell proteins were collected for western blot. GAA vs. Control: p = 0.0288; GAA + Recilisib vs. GAA: p = 0.0004; n = 3. ( G ) BV2 cells were treated with GAA (20 µM), recilisib (20 µM) and EACC (2 µM), in the presence of Aβ42 (2 µM) for 24 h, and intracellular Aβ42 levels were measured by ELISA. DMSO at 0.1% was used as the control. GAA vs. Control: p = 0.0031; GAA + EACC vs. GAA: p = 0.0064; n = 3. n is the number of replicates (biological and technical) used for each of the described results. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. indicated control.

Article Snippet: The human Aβ42 enzyme-linked immunosorbent assay (ELISA) kit was purchase from Cusabio (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer’s Disease (Mouse Model) through Autophagy Induced by Activating Axl

doi: 10.3390/ijms22115559

Figure Lengend Snippet: GAA activates the autophagy pathway through Axl. BV2 cells were ( A ) treated with GAA at the indicated concentrations for 6 h or ( B ) treated with GAA (20 µM) for the indicated periods of time, and whole cell proteins were collected for Western blot. ( A ) GAA-5 vs. DMSO: p = 0.0452; GAA-20 vs. DMSO: p = 0.0034; n = 3. ( B ) 6 h vs. 0 h: p = 0.0299; 12 h vs. 0 h: p = 0.0073; n = 3. ( C – F ) BV2 cells were pretreated with 0.1% DMSO or the indicated Axl antagonist (R428 at 5 µM) for 30 min, followed by administration of GAA (20 µM) and Aβ42 (2 µM) for 6 h. Whole cell proteins then were collected for Western blot. ( D ) Aβ42 + GAA vs. Aβ42: p <0.0001; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p < 0.0001; n = 3. ( E ) Aβ42 + GAA vs. Aβ42: p = 0.028; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0304; n = 3. ( F ) Aβ42 + GAA vs. Aβ42: p = 0.0247; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0009; n = 3. ( G – H ) BV2 cells were pretreated with R428 (5 µM) for 30 min, followed by administration of FITC-Aβ42 (1 μM) and GAA (20 µM) for 6 h, and cells were immunostained with LysoTracker Red. Representative images show the co-localization of Aβ42 and lysosomes (yellow). Scale bar: 50 µm. Aβ42 + GAA vs. Aβ42: p = 0.0469; Aβ42 + GAA + R428 vs. Aβ42 + GAA p = 0.0057; n = 4. ( I ) BV2 cells were pretreated with the indicated antagonists of Axl (R428 at 5 µM) and Pak1 (IPA-3 at 20 µM), followed by administration of GAA (20 µM) and Aβ42 (2 µM) for 24 h. The intracellular Aβ42 levels were measured by ELISA. DMSO at 0.1% was used as the control. GAA vs. Control: p = 0.0012; GAA + R428 vs. GAA: p = 0.0151; GAA + IPA vs. GAA: p = 0.0015; n = 3. n is the number of replicates (biological and technical) used for each of the described results. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. indicated control.

Article Snippet: The human Aβ42 enzyme-linked immunosorbent assay (ELISA) kit was purchase from Cusabio (Wuhan, China).

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

Journal: International Journal of Molecular Sciences

Article Title: Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer’s Disease (Mouse Model) through Autophagy Induced by Activating Axl

doi: 10.3390/ijms22115559

Figure Lengend Snippet: GAA ameliorates Aβ42-induced behavioral deficits in Aβ-injected mice through Axl. ( A ) Experimental design for the animal study. Cerebroventricular injection of aggregated Aβ42 (82 pmol/µL, 5 µL/mouse) was applied to 8-week-old mice. ( B ) Representative motion track and ( C ) the discrimination index in the object recognition test. Aβ42 vs. Sham: p = 0.0019; Aβ42 + Rog vs. Aβ42: p = 0.0101; Aβ42 + GAA vs. Aβ42: p = 0.0038; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0078; n = 8 ( D ) Escape latency during spatial-acquisition training. (Day2) Aβ42 vs. Sham: p = 0.0011; Aβ42 + Rog vs. Aβ42: p = 0.0303; Aβ42 + GAA vs. Aβ42: p = 0.0304; n = 8. (Day3) Aβ42 vs. Sham: p = 0.0002; Aβ42 + Rog vs. Aβ42: p = 0.0019; Aβ42 + GAA vs. Aβ42: p = 0.0028; n = 8. (Day4) Aβ42 vs. Sham: p < 0.0001; Aβ42 + Rog vs. Aβ42: p < 0.0001; Aβ42 + GAA vs. Aβ42: p < 0.0001; n = 8. (Day5) Aβ42 vs. Sham: p = 0.0004; Aβ42 + Rog vs. Aβ42: p = 0.0002; Aβ42 + GAA vs. Aβ42: p = 0.0002; n = 8. ( E ) Representative motion track, ( F ) the platform-crossing number, (Aβ42 vs. Sham: p < 0.0001; Aβ42 + Rog vs. Aβ42: p < 0.0001; Aβ42 + GAA vs. Aβ42: p < 0.0001; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p < 0.0001; n = 8.) ( G ) distance in the target quadrant (Aβ42 vs. Sham: p < 0.0001; Aβ42 + Rog vs. Aβ42: p = 0.0004; Aβ42 + GAA vs. Aβ42 p < 0.0001; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0002; n = 8.) and ( H ) time spent in the target quadrant (Aβ42 vs. Sham: p = 0.0002; Aβ42 + Rog vs. Aβ42: p = 0.0002; Aβ42 + GAA vs. Aβ42: p =0.0003; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0001; n = 8.) in the spatial-probe test. ( I ) Aβ42 level in the hippocampus detected by ELISA. Aβ42 vs. Sham: p = 0.0014; Aβ42 + Rog vs. Aβ42: p = 0.0064; Aβ42 + GAA vs. Aβ42: p = 0.0266; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0053; n = 8. ( J ) LC3B level in the hippocampus detected by IHC assessment. Images were obtained under a microscope (scale bar: 100 μm). Aβ42 + GAA vs. Aβ42: p = 0.0004; Aβ42 + GAA + R428 vs. Aβ42 + GAA: p = 0.0009; n = 4. n is the number of replicates (biological and technical) used for each of the described results. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. Sham group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Aβ42 group ( n = 8 mice/ group).

Article Snippet: The human Aβ42 enzyme-linked immunosorbent assay (ELISA) kit was purchase from Cusabio (Wuhan, China).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: PLoS ONE

Article Title: HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

doi: 10.1371/journal.pone.0077972

Figure Lengend Snippet: ( A ) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). ( B ) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. ( C ) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. ( D ) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. ( E ) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. ( F ) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.

Article Snippet: Purified recombinant APP (cat. no APP-526H, Creative BioMart, NY, USA) was resuspended in PBS supplemented with 1% NP-40 to yield a final concentration of 1 ng/μl, and 500 μl was incubated with GST- or GST-Tat-coated beads for 3 hours at 4°C.

Techniques: Purification, SDS Page, Staining, GST Pulldown Assay, Incubation, Western Blot, Transfection, Transduction, Virus, Recombinant, Produced