Journal: The Journal of Neuroscience

Article Title: Neuroprotective Effects of Regulators of the Glycogen Synthase Kinase-3β Signaling Pathway in a Transgenic Model of Alzheimer's Disease Are Associated with Reduced Amyloid Precursor Protein Phosphorylation

doi: 10.1523/JNEUROSCI.4321-06.2007

Figure Lengend Snippet: Effects of lithium on GSK3β activity and APP metabolism in hAPP tg mice. A, Levels of GSK3β activity, expressed as counts per minute (CPM) per microgram of protein, in homogenates from the frontal cortex of nontg and hAPP tg mice treated with saline or lithium. B, Aβ1–42 levels in homogenates from the frontal cortex of nontg and hAPP tg mice treated with saline or lithium. C, Immunoreactivity levels of the Aβ-cleaving enzyme neprilysin in homogenates from the frontal cortex of nontg and hAPP tg mice treated with saline or lithium. D, Levels of BACE1 immunoreactivity in homogenates from the frontal cortex of nontg and hAPP tg mice treated with saline or lithium. *p < 0.05 compared with saline-treated hAPP tg mice by one-way ANOVA with post hoc Tukey–Kramer test; n = 6 mice per group.

Article Snippet: Blots were incubated with antibodies against full-length (FL) APP (mouse monoclonal, clone 22C11, 1:20,000; Chemicon, Temecula, CA), Aβ (mouse monoclonal, clone 6E10, 1:1000; Signet Laboratories, Dedham, MA), APP C-terminal fragments (CTFs) (rabbit polyclonal CT15, 1:2500; courtesy of Dr. E. Koo, University of California, San Diego, La Jolla, CA), phosphorylated APP (APP-p) (Thr668, 1:1200; Cell Signaling Technology, Beverly, MA), neprilysin (mouse monoclonal, clone CD10, 1:1000; Abcam, Cambridge, MA), or β-secretase 1 (BACE1) (1:1000; ProSci, Poway, CA), followed by secondary antibodies tagged with horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) and were visualized by enhanced chemiluminescence and analyzed with a Versadoc XL imaging apparatus (Bio-Rad, Hercules, CA).

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: Neuroprotective Effects of Regulators of the Glycogen Synthase Kinase-3β Signaling Pathway in a Transgenic Model of Alzheimer's Disease Are Associated with Reduced Amyloid Precursor Protein Phosphorylation

doi: 10.1523/JNEUROSCI.4321-06.2007

Figure Lengend Snippet: GSK3β activity and APP metabolism in hAPP/DN–GSK3β double tg mice. A, Levels of GSK3β activity in immunoprecipitated samples from the frontal cortex of nontg, hAPP tg, DN–GSK3β tg, and hAPP/DN–GSK3β double tg mice. B, Levels of GSK3β activity in PP1γ-treated immunoprecipitated samples from the frontal cortex of nontg and DN–GSK3β tg mice. C, Semiquantitative immunoblot analysis of levels of GSK3β-p in immunoprecipitated samples from the frontal cortex of nontg and DN–GSK3β tg mice. D, Aβ1–42 levels in homogenates from the frontal cortex of nontg, hAPP tg, DN–GSK3β tg, and hAPP/DN–GSK3β tg mice. E, Immunoreactivity levels of the Aβ-cleaving enzyme neprilysin in homogenates from the frontal cortex of nontg, hAPP tg, DN–GSK3β tg, and hAPP/DN–GSK3β tg mice. F, Levels of BACE1 immunoreactivity in homogenates from the frontal cortex of nontg, hAPP tg, DN–GSK3β tg, and hAPP/DN–GSK3β tg mice. *p < 0.05 compared with hAPP tg mice by unpaired two-tailed Student's t test; n = 6 mice per group. **p < 0.05 compared with samples that were not treated with PP1γ by unpaired two-tailed Student's t test; n = 6 mice per group.

Article Snippet: Blots were incubated with antibodies against full-length (FL) APP (mouse monoclonal, clone 22C11, 1:20,000; Chemicon, Temecula, CA), Aβ (mouse monoclonal, clone 6E10, 1:1000; Signet Laboratories, Dedham, MA), APP C-terminal fragments (CTFs) (rabbit polyclonal CT15, 1:2500; courtesy of Dr. E. Koo, University of California, San Diego, La Jolla, CA), phosphorylated APP (APP-p) (Thr668, 1:1200; Cell Signaling Technology, Beverly, MA), neprilysin (mouse monoclonal, clone CD10, 1:1000; Abcam, Cambridge, MA), or β-secretase 1 (BACE1) (1:1000; ProSci, Poway, CA), followed by secondary antibodies tagged with horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) and were visualized by enhanced chemiluminescence and analyzed with a Versadoc XL imaging apparatus (Bio-Rad, Hercules, CA).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Two Tailed Test

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: The Journal of Neuroscience

Article Title: Complete Rescue of Cerebrovascular Function in Aged Alzheimer's Disease Transgenic Mice by Antioxidants and Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist

doi: 10.1523/JNEUROSCI.3348-08.2008

Figure Lengend Snippet: Improved cerebrovascular reactivity coincides with alterations in oxidative stress regulation. A, The responses of isolated arteries to ACh, CGRP, and NOS inhibition with l-NNA (10−5 m) of aged APP mice (▲) relative to wild-type (●) littermates (★p < 0.05, ★★p < 0.01, ★★★p < 0.001) were completely normalized in tempol-, NAC-, and pioglitazone (pio)-treated APP (△) mice, whereas they were unaffected in treated wild-type (○) mice (untreated vs treated APP mice, *p < 0.05, **p < 0.01, ***p < 0.001; n = 4–8 mice/group). B, Pioglitazone, but not NAC or tempol (data not shown), reversed the SOD2 upregulation detected by Western blot in pial vessels of APP mice. Actin was used to normalize loading variation (n = 5–7 mice/group). C, All compounds attenuated the increased SOD2 immunoreactivity detected throughout the cortical neuropil of APP mice, but not the bulk of enzyme immunointensity in neuronal cell bodies (n = 3–5 mice/group). Scale bar, 20 μm. Error bars represent SEM.

Article Snippet: Membranes loaded with proteins (4–20 μg) were incubated with either rabbit anti-SOD1 [1:3000 (vessels)/1:10,000 (cortex); Stressgen], anti-SOD2 (1:20,000/1:10,000; Stressgen), anti-APP (1:2000/1:500; ProSci), anti-BACE1 (1:1000, Santa Cruz Biotechnology), mouse anti-endothelial NOS (eNOS; 1:500; BD Biosciences Transduction Laboratories), anti-cyclooxygenase-2 (COX-2; 1:200/1:100; Cayman), anti-p67 phox (1:200; BD Biosciences Transduction Laboratories), anti-Aβ (6E10, 1:1000/1:200; BioSource International), or anti-actin (1:10,000, Sigma), which was used to normalize loading variation.

Techniques: Isolation, Inhibition, Western Blot

Journal: The Journal of Neuroscience

Article Title: Complete Rescue of Cerebrovascular Function in Aged Alzheimer's Disease Transgenic Mice by Antioxidants and Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist

doi: 10.1523/JNEUROSCI.3348-08.2008

Figure Lengend Snippet: Treatment effect on amyloidosis. A, APP mice had increased levels of APP in pial vessels (★★★p < 0.001) and cortex (data not shown), although levels of BACE1 were unchanged. They also displayed increased total soluble Aβ, as well as soluble and insoluble Aβ1–40 and Aβ1–42. Neither NAC (data not shown) nor pioglitazone (pio) had an effect on APP, its cleavage enzyme BACE1, or cleavage product Aβ. Actin was used to normalize loading variation (n = 5–7 mice/group). Sol, Soluble; white bars, wild type (WT); light gray bars, WT plus pio; black bars, APP; dark gray bars, APP plus pio. B, Aged APP mice featured extensive Aβ plaque deposition in the cortex and hippocampus (Hi), as evidenced by thioflavine S (left) and Aβ1–42 immunostaining (right), compared with wild-type littermates, which had no deposition (data not shown). The treatments did not affect Aβ plaque number or load (n = 5–6 mice/group). Scale bar, 300 μm. Error bars represent SEM.

Article Snippet: Membranes loaded with proteins (4–20 μg) were incubated with either rabbit anti-SOD1 [1:3000 (vessels)/1:10,000 (cortex); Stressgen], anti-SOD2 (1:20,000/1:10,000; Stressgen), anti-APP (1:2000/1:500; ProSci), anti-BACE1 (1:1000, Santa Cruz Biotechnology), mouse anti-endothelial NOS (eNOS; 1:500; BD Biosciences Transduction Laboratories), anti-cyclooxygenase-2 (COX-2; 1:200/1:100; Cayman), anti-p67 phox (1:200; BD Biosciences Transduction Laboratories), anti-Aβ (6E10, 1:1000/1:200; BioSource International), or anti-actin (1:10,000, Sigma), which was used to normalize loading variation.

Techniques: Immunostaining