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: bioRxiv

Article Title: Lysosomal Exocytosis Releases Pathogenic α-Synuclein Species from Neurons

doi: 10.1101/2021.04.10.439302

Figure Lengend Snippet: ( a-c ) Recombinant purified myc-αSyn was shaken at 37°C in presence of concentrated extracellular medium from mouse cortical neuron cultures collected over 7 days (DIV 42-49), generated either from wild type (WT) mice, or from Tg x2 -αSyn A53T mice with or without lentiviral expression of VAMP7 dominant-negative fragment (VAMP7 DN ; infected at DIV 7). Aggregation of myc-αSyn was analyzed at the indicated days of incubation by the following assays: ( a ) Congo-red derivative, amyloid-binding dye K114 fluorescence at 390/535 nm (n=4). ( b ) Amyloid-binding dye Thioflavin-T fluorescence at 450/485 nm (n=4). ( c ) Quantitative immunoblotting for the myc epitope-tag, where aggregation is measured as disappearance of monomeric myc-αSyn (top; n=4); dot-blotting for filamentous myc-αSyn aggregates using αSyn Fila antibody (middle; n=4); and dot-blotting for amyloid-type myc-αSyn aggregates using αSyn Amyl A11 antibody (bottom; n=4). All data represent means ± SEM, where each ‘n’ is an independent aggregation experiment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by RM 2-way ANOVA.

Article Snippet: β-Actin: Sigma (A1978); A11 oligomers: Stressmarq (SPC-506D); ATP5G: Abcam (ab181243); Calreticulin: Thermo Fisher (OTI15F5) and Novus (NB600-103); Cathepsin-L: Novus (JM10-78); CD81: Novus (SN206-01); EEA1: Thermo Fisher (MA5-14794); Flag: Sigma Cl.

Techniques: Recombinant, Purification, Generated, Expressing, Dominant Negative Mutation, Infection, Incubation, Binding Assay, Fluorescence, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Adapentpronitrile, a New Dipeptidyl Peptidase-IV Inhibitor, Ameliorates Diabetic Neuronal Injury Through Inhibiting Mitochondria-Related Oxidative Stress and Apoptosis

doi: 10.3389/fncel.2018.00214

Figure Lengend Snippet: List of primary and secondary antibodies used in the study.

Article Snippet: Membranes were probed overnight at 4°C with primary antibodies for specific detection of APP (dilution 1:500; Boster, China, BA0581), Aβ (dilution 1:1000; Abcam, United Kingdom, ab62658), GLP-1R (dilution 1:500, Bioss, China, bs-1559R), Bcl-2 (dilution 1:500; Abcam, United Kingdom, ab196495), Bax (dilution 1:400; Proteintech, China, 50599-2-Ig), cytochrome c (dilution 1:1000; Abcam, United Kingdom, ab133504), caspase-9 (dilution 1:1000; Abcam, United Kingdom, ab184786), caspase-3 (dilution 1:1000; Abcam, United Kingdom, ab184787), and β-actin (dilution 1:3000; Proteintech, United States, 60008-1-Ig; Table ).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Adapentpronitrile, a New Dipeptidyl Peptidase-IV Inhibitor, Ameliorates Diabetic Neuronal Injury Through Inhibiting Mitochondria-Related Oxidative Stress and Apoptosis

doi: 10.3389/fncel.2018.00214

Figure Lengend Snippet: Effects of adapentpronitrile on pathomorphology and the expression of APP and Aβ proteins in hippocampus and cortex of rat. (A) Representative neuronal pathomorphology in hippocampus and cortex. Sections were pictured at 400×. Scale bar = 50 μm. (B) The expressions of APP and Aβ proteins were measured by Western Blotting. Data were expressed as mean ± SD of four independent experiments, and were analyzed statistically using one-way ANOVA, followed by Dunnett-t type multiple comparison tests. # P < 0.05 and ## P < 0.01 vs. Control group, respectively; ∗ P < 0.05 and ∗∗ P < 0.01 vs. HFD/STZ group, respectively.

Article Snippet: Membranes were probed overnight at 4°C with primary antibodies for specific detection of APP (dilution 1:500; Boster, China, BA0581), Aβ (dilution 1:1000; Abcam, United Kingdom, ab62658), GLP-1R (dilution 1:500, Bioss, China, bs-1559R), Bcl-2 (dilution 1:500; Abcam, United Kingdom, ab196495), Bax (dilution 1:400; Proteintech, China, 50599-2-Ig), cytochrome c (dilution 1:1000; Abcam, United Kingdom, ab133504), caspase-9 (dilution 1:1000; Abcam, United Kingdom, ab184786), caspase-3 (dilution 1:1000; Abcam, United Kingdom, ab184787), and β-actin (dilution 1:3000; Proteintech, United States, 60008-1-Ig; Table ).

Techniques: Expressing, Western Blot, Comparison, Control